Trypanosoma cruzi: Early parasite proliferation and host resistance in inbred strains of mice

EXPERIMENTAL

62, 194-201 (1986)

PARASITOLOGY

Trypanosoma

cruzi:

Early Parasite Proliferation in Inbred Strains of Mice

and Host Resistance

THOMAS M. TRISCHMANN Department

of Immunology

and Infectious Diseases, School of Hygiene and Public Health, University, Baltimore, Maryland 21205, U.S.A.

Johns Hopkins

(Accepted for publication 5 March 1986) TRISCHMANN, T. M. 1986. Trypanosoma cruzi: Early parasite proliferation and host resistance in inbred strains of mice. Experimental Parasitology 62, 191-201. The extent of parasite proliferation following completion of the first cycle of intracellular replication was significantly higher in CD-l nulnu mice and in irradiated mice compared to other, including highly susceptible, mouse strains. A control of parasite proliferation thus occurs in normal mice as early as the first cycle of intracellular replication. The thymus dependency and radiation sensitivity of the early control of proliferation of Trypanosoma cruzi suggest that an immune response to the parasite is involved in the early control of proliferation. The BXH-2 recombinant inbred strain demonstrated an inability to control early proliferation and, 4-5 days after infection, had parasitemias several times higher than those observed in susceptible mouse strains. The BXH-2 strain appears to lack the early control mechanism. When the extent of proliferation of T. cruzi at completion of the first cycle of intracellular replication was compared in inbred strains of mice having varying levels of resistance to the parasite, the extent of proliferation correlated with host resistance, being lowest in the most resistant strains (C57BLi6, SJL) and highest in the most susceptible strains (C3H, A). It is suggested that the mechanism(s) controlling early parasite proliferation may be of primary importance as the basis for host resistance. 0 1986 Academic Press, Inc. INDEX DESCRI~ORS: Trypanosoma cruzi; Protozoa, parasitic; Hemoflagellate; Chagas’ disease; Mouse, inbred; Natural resistance; Natural killer (NK) cell; Interferon.

of proliferation of an organism may vary greatly in different inbred strains of mice A genetic basis for resistance to Trypano- and that the differing rates of proliferation soma cruzi in inbred strains of mice has can be demonstrated within a few days been well established (Pizzi et al. 1949; after infection (Hirst and Wallace 1976; Trischmann et aZ. 1978). The mechanism(s) Bradley 1979; Cheers et al. 1978; Horthrough which this genetic basis acts to in- maeche 1979; Albright and Albright 1981). fluence host resistance has not been identi- An early control of proliferation of T. cruzi fied. Recognition of the importance of the occurs in inbred strains of mice in the first 2 immune response for host survival has led weeks after infection with the Brazil strain to comparisons of specific and nonspecific of the parasite (Trischmann 1983). Parasite immune responses in resistant and suscep- levels in the blood during this time are nortible strains of mice (Nogueira and Cohn mally too low to allow comparative assess1976; Powell and Kuhn 1980; Hatcher et al. ment of parasitemia levels in different 1981; Trischmann 1983; Lalonde et al. mouse strains. The higher parasitemias 1985). No aspect of the parasite specific usually seen later in the acute phase in susimmune response, however, has been ceptible mouse strains compared to resisfound to correlate with host resistance. tant strains could be due to differences in Studies involving other parasites and the immune responses occurring during bacteria have shown that the early extent this period or to differences in the ability to 194 0014-4894/86$3.00 Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

Trypanosoma

cruzi: RE~ISTANCEININBREDMICE

19.5

tion, mice of a particular inbred strain, along with a control group of C3H mice, were injected with 20 x 106 trypomastigotes. Since mice did not synchronously reach maximum parasitemias, parasitemias were measured at 6 hr intervals beginning at 96 hr after challenge and continuing until a decrease in parasitemia was observed. The highest parasitemia attained by each mouse was used in calculating the mean maximum parasitemia for the group. The ratio of the mean for the experimental group to the mean for the control C3H group was then determined. Thus, MATERIALSANDMETHODS the extent of parasite proliferation in any strain was CD-l nulnu mice and heterozygous littermates were always measured relative to C3H mice. purchased from Charles River Laboratories Interferon levels were quantified by means of a cy(Kingston, NY, USA). All other inbred mouse strains topathic effect inhibition assay (Armstrong 1981)using were obtained from the Jackson Laboratory (Bar the Indiana strain of vesicular stomatitis virus and Harbor, ME, USA). F, and F, progeny were bred in murine L-929 cells. The interferon titer was defined as our animal facilities. Female mice, 8 to 10 weeks old, the reciprocal of the serum dilution that reduced cytowere used in all experiments. pathology by 50%. Interferon titers are expressed in Irradiated mice received 900 rads from a 13’Cs international units. A U.S. National Institutes of source at a rate of 120 radsimin. Irradiated mice were Health G-002-904-511 reference standard was included maintained on acidified water. in all assays. The Brazil strain of Trypan~~~ma cruzi was used in The Mann-Whitney test was used to determine the all experiments and was maintained by passage in significance of differences between experimental C3HIHeJ mice. Challenge with bloodstream trypogroups. mastigotes and determination of blood parasitemias RESULTS were performed as described previously (Trischmann Intravenous injection of 20 x lo6 Try1983). To obtain large numbers of parasites, trypomastipanosoma cruzi trypomastigotes into C3H gotes were harvested from infected Vero cell cultures. mice resulted in a blood parasitemia of Vero cells were grown to confluence in tissue culture flasks and initially inoculated with blood drawn ster- about 10 x IO6parasites/ml 15 min after inilely from the heart of an infected mouse. Cultures jection. The level of blood parasitemia decreased over the following 24 hr to l-2 x were maintained at 37 C in a humidified 5% CO, atmosphere in Dulbecco’s modified Eagle’s medium conlo6 parasites/ml. Upon completion of the taining 2% fetal bovine serum, 100 IU penicillin/ml, first cycle of intracellular replication, 100 kg streptomycin/ml, and 2 mM L-glutamine (all which took about 4 days, a sharp increase from Grand Island Biological Co., Grand Island, NY, in blood parasitemia occurred which was USA). Trypomastigotes harvested following the initial followed a day later by a more gradual deblood inoculation were frozen in Dulbecco’s modified Eagle’s medium containing 10% glycerol and 10% crease in parasite numbers (Fig. 1). The fetal bovine serum and stored in liquid nitrogen. maximum parasitemia seen during this peEvery 2 months, a vial of the original stock was riod occurred about 4% days after the time thawed and used to initiate a new infection. Initial cell of infection. lysis and release of parasites generally occurred 4 to 5 The extent of parasite proliferation foldays after infection. Trypomastigotes harvested from the culture fluid were used to infect new flasks of cell lowing the first cycle of intracellular replicultures. One day after transfer of parasites to a new cation was compared for a series of inbred flask, the medium was changed to remove extracelmouse strains varying in their level of resislular parasites. tance to T. cruzi (Table I). The extent of For injection into mice, cell culture fluid containing parasite proliferation in each strain was trypomastigotes was centrifuged at 15OOgfor 30 min. Trypomastigotes were brought to a concentration of measured relative to the extent of prolifera108/mlin serum-free Dulbecco’s modified Eagle’s me- tion seen in susceptible C3H mice as dedium. Mice were injected intravenously with 0.2 ml scribed under Materials and Methods. Par(20 x lo6 parasites). asite proliferation in a second susceptible To determine the extent of parasite proliferation at completion of the first cycle of intracellular replicastrain (A) was slightly greater than that

limit parasite proliferation early in the infection. In the present. study, intravenous injection of large numbers of parasites is used to investigate how soon after infection this control of proliferation of T. cvuzi occurs and if differences in extent of early parasite proliferation are related to host resistance.

196

THOMAS

M. TRISCHMANN

16-

LI

1111

I1

123456769

Days

After

I

II

Infection

1. Course of parasitemia in C3H mice (10 mice) injected intravenously with 20 x lo6 trypomastigotes of T~~uzosomu cruzi. Each point represents the mean k SD. FIG.

found in C3H mice. Proliferation was lowest in the two most resistant strains tested (C57BL/6 and SJL) and of an intermediate degree in strains showing moderate levels of resistance (AKR and BALB/ c). Proliferation was also low in (C3H x C57BLi6)Fi mice which are highly resistant and in C57BL/6-bglbg mice which carry the beige mutation. The most extensive proliferation occurred in two inbred strains (BXH-2 and CD-l nulnu) known to lack an early control of parasite proliferation as measured 16 days after infection (Trischmann 1983) and in irradiated mice. For 3 days after injection, lethally irradiated mice did not differ significantly in their levels of parasitemia compared to those observed in mm-radiated C3H mice (Fig. 2). By Day 4, however, parasitemias were significantly higher (P < 0.001) in irradiated mice. The extent of proliferation in irradiated mice was similar in mice irra-

diated 1 or 2 days prior to challenge (Fig. 3a). When mice were irradiated after challenge, the degree of proliferation decreased as irradiation was administered at later times (Fig. 3b). Irradiation 24 hr after infection had an effect similar to irradiation before challenge, while irradiation 72 hr after infection did not lead to a significant increase in the extent of parasite proliferation. The maximum mean parasitemia was significantly (P < 0.001) higher compared to unirradiated control mice in all irradiated groups except for mice irradiated 3 days after infection (Fig. 3b). C3H and F, mice derived from crosses between (C57BL/6 x BXH-2)Fi mice were injected with 20 x lo6 parasites. Parasitemias were determined 4-5 days later, and mice were classified as either having parasitemias similar to those seen in BXH-2 mice or lower than seen in the C3H controls. Of 56 F, mice tested, 15 (27%) had very high parasitemia levels at completion of the first cycle of replication (data not shown). Serum interferon levels were measured in C3H, C57BL/6, BXH-2, and irradiated C3H mice 24 hr after challenge with lo4 blood derived trypomastigotes. The interferon levels in sera from C3H mice were slightly but consistently higher than those found in C57BL/6 mice (Table II). BXH-2 recombinant inbred mice and irradiated mice had no detectable serum levels of interferon. DISCUSSION

We had noted previously that an early control of proliferation of Trypanosoma cruzi occurs in inbred strains of mice. The early control was effective during the first 2 weeks following infection with lo4 parasites of the Brazil strain. Intravenous inoculation of a large number of trypomastigotes has allowed us to follow parasite proliferation during the first cycle of intracellular replication, which takes approximately 4 days in the mouse (Howells

Trypanosoma

197

cruzi: RESISTANCE IN INBRED MICE

TABLE I Relative Maximum Blood Parasitemias in Inbred Strains of Mice following the First Cycle of Intracellular Replication of Trypanosoma cruzP Mouse strain C3HIHe (irradiated)d C57BLl6 (irradiated)d BXH-2 CD-l nuinu CD-l nul+ A C3HIHe AKR BALBic SJL C57BLl6 (C3H x C57BL/6)F, C57BW6-bgibg

Level of resistanceb Low Low Low Low Low Low Intermediate Intermediate High High High High

Ratio of maximum mean parasitemias Experimental mice:C3H controlsC 4.37 3.84 3.48 2.67 2.49 3.06 2.03 1.65 0.93 1.24 1.28 1.00 0.74 0.56 0.71 0.87 0.32 0.53 0.28 0.41 0.33 0.33

a Parasitemias were measured at 6 hr intervals from Day 4 to 5 after intravenous injection of 20 x lo6 cell culture derived trypomastigotes. The results from two separate experiments are usually shown for each mouse strain. b Resistance was determined by survival of mice 8 weeks after challenge with lo4 trypomastigotes of the Brazil strain of T. cruzi. c Each experimental group contained 10 to 12 mice. Each control group of C3H mice contained at least 5 mice. d Mice received 900 rads 24 hr prior to injection of parasites.

and Chiari 1975). The extent of parasite proliferation at completion of the first cycle of intracellular replication was significantly higher in CD-I nulnu mice and in irradiated mice compared to the highly susceptible C3H strain. A control of parasite proliferation thus occurs in normal mice as early as the first cycle of intracellular replication. The BXH-2 recombinant inbred strain was the only inbred strain other than nulnu mice to have previously exhibited a lack of early control of parasite proliferation (Trischmann 1983). In the present study, BXH-2 mice again demonstrated an inability to control early proliferation as evidenced by their having parasitemias several times higher than those observed in susceptible mouse strains at 4-5 days after infection. When the extent of proliferation of T. cmzi following completion of the first cycle of intracellular replication was compared

between inbred strains of mice having varying levels of resistance to the parasite, the extent of proliferation correlated with host resistance, being lowest in the most resistant strains (C57BLi6, SJL) and highest in the most susceptible strains (C3H, A). This type of comparison was not possible in our previous studies where survival was assessedfollowing challenge with IO4 parasites. This challenge dose resulted in parasitemias still too low after 2 weeks to allow for an accurate comparison of parasite levels in different mouse strains. Parasite levels at later times are high enough for accurate quantitation but are likely to be strongly influenced by the developing antibody response to the parasite (Trischmann 1983). More sensitive methods for quantitating blood parasitemias are being developed to allow us to follow early parasite levels after challenge with low numbers of parasites.

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fection was lethal for all strains. Nevertheless, C57BL/6 mice were in the low parasitemia group while C3H, A, and BALB/c mice were in the high parasitemia 60 group. It appears likely that the defect in BXH-2 mice which leads to the inability to control proliferation at Day 16 after infection is also responsible for the high parasite levels observed by Day 5 in this present study. The high parasitemia seen in BXH-2 mice 16 days after infection is the result of a mutation at a single locus which may have occurred in the inbreeding of the strain (Trischmann 1984). Consequently, 25% of F, mice derived from matings of (C57BL/6 x BXH-2)F, mice show the high parasitemia trait when screened 16 days after infection. A group of F, mice was tested for the extent of proliferation 4-5 days after Days After Infection challenge with 20 x lo6 parasites, and 27% FIG. 2. Course of parasitemia in C3H mice (A), ir- again displayed a lack of control of early radiated (900 rads) C3H mice (O), and irradiated (900 parasite proliferation. The BXH-2 strain rads) C57BL/6 mice (4) following intravenous injecshould prove to be of especial use for study tion of 20 x lo6 trypomastigotes of Trypanosoma cuuzi. Each point represents the mean ? SD. The of the mechanism involved in early control C3H control group consisted of 6 mice and each of the of parasite proliferation. irradiated groups consisted of 10 mice. Control of parasite proliferation during the first intracellular cycle of replication could be effected during several different periods. Parasites could be acted upon by Varying degrees of parasite proliferation in different mouse strains were also noted the host prior to their invasion of cells, by Wrightsman et al. (1982) in their studies during the intracellular period of replicaof the genetic control of responses to T. tion, or following their release from cells. cruzi in mice. They compared the levels of About 90% of the parasites initially found in the blood after intravenous injection of parasitemia reached in various inbred mouse strains 17 days after infection with 20 x lo6 trypomastigotes are cleared from the Peru strain of T. cruzi. The exceptional the blood in 24 hr. The main mechanism for virulence of the Peru strain was evident by control of proliferation does not appear to the attainment in some mouse strains of be effected during this period since irradiaparasitemias in excess of 10’ parasites/ml tion of mice 24 hr after infection leads to as only 17 days after challenge with lo3 para- great a degree of proliferation as irradiation sites. Other mouse strains had lower para- prior to infection. If control was primarily sitemias at this time, and it was suggested effected during the first 24 hr, irradiation that inbred strains could be divided into after this time would not affect the control two groups, high parasitemia strains and mechanism. The failure of irradiation, at 72 low parasitemia strains. The relative resis- hr after infection, to exacerbate the extent tance of the strains tested, based on sur- of proliferation does not eliminate the posvival, could not be established since the in- sibility of a destruction of emerging para7ot

I

Trypanosoma

199

cruzi: RESISTANCE IN INBREDMICE

60-

T T

48 hrs

-24 hrs Time

of Irradiation

+24hr

Relolive

lo Time

s

+48hr!

_I + 72 hrs

of Challenge

3. Maximum parasitemias attained in C3H mice irradiated (900 rads) before (a) or after (b) intravenous injection with 20 x lo6 trypomastigotes of Trypunosomu cruzi. The mean L SD is shown for each group. Each group in (a) consisted of 8 mice while each group in (b) consisted of 7 mice. FIG.

fection with T. cruzi wherein the extent of early parasite proliferation becomes a major determinant of the outcome of the infection. The development of a high parasitemia late in the acute phase, which usually occurs in a susceptible mouse strain, may be more the result of previous extensive proliferation of the parasite than the result of some deficiency in the immune response occurring during the late acute phase. Thus, comparison of the immune responses of resistant and susceptible strains late in the acute phase may not reTABLE11 veal differences of particular relevance to Serum Interferon Titers 24 hr after Infection with lo4 the genetic basis of host resistance, alBloodstream Trypomastigotes of Trypnnosoma cruzi though such differences could have a secNumber of International ondary influence on resistance. Rather, the Mousestrain mice units/ml mechanism(s) controlling early parasite C3HlHe 7 1675 proliferation may be of primary importance C57BLl6 5 749 BXH-2 5 <20 as the basis for host resistance. Such C3HIHe (irradiatedP 5 <20 “early” and “late” factors affecting the LiMice received900rads 24 hr prior to infection. outcome of an infection in mice have been

sites since the effects of the irradiation may not be immediate. A comparative study of tissue parasite loads in different mouse strains during the first cycle of replication should help in determining when control is effected. For many infectious organisms, survival of the host is dependent upon an effective immune response being mounted before extensive proliferation of the organism can occur. A similar situation may exist for in-

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THOMASM.TRISCHMANN

described for Salmonella typhimurium (Hormaeche 1979) and Leishmania donovani (Blackwell et al. 1980). There is limited information concerning immune responses to T. cruzi very early in the course of infection. Increases in NK cell activity (Hatcher et al. 1981) and in serum interferon levels (Sonnenfeld and Kierszenbaum 1981) do occur within a few days after infection with T. cruzi. A role for NK cells in the early control of proliferation is not suggested by our data. The SJL and C57BL/6-bglbg strains are deficient in endogenous and inducible NK cell activity (Kaminsky et al. 1983; Roder 1979), but both displayed a low degree of early proliferation of T. cruzi during the first cycle of replication. Hatcher et al. (1981) have reported that the resistant C57BL/6 strain and the susceptible C3H strain showed comparable increases in NK cell activity following infection. Quan et al. (1983), using an anti-interferon antiserum to diminish NK cell activity in C57BL/6 mice, found no enhancement of parasitemia in these mice compared to control mice. The role of interferon in protection against T. cruzi remains unclear. Administration of interferon to mice immediately prior to and for 6 days after infection with T. cruzi increased their level of resistance as indicated by decreased levels of parasitemia (Kierszenbaum and Sonnenfeld 1982). In contrast, earlier studies failed to find any protective effect of interferon or found that interferon inducers actually exacerbated the course of infection (Brener 1980). We did not find a lower serum interferon level in C3H mice compared to C57BL/6 mice 24 hr after infection as would be expected if the early production of interferon was associated with the differing extents of parasite proliferation in these strains. However, the BXH-2 strain was notable for its lack of any detectable serum interferon response. Whether this failure to produce interferon is related to the absence of an early control of proliferation in this strain will need to be

determined by following both traits in a series of genetic crosses. The 24 hr interferon response was also absent in lethally irradiated mice, which had a degree of parasite proliferation comparable to that found for BXH-2 mice. It is possible that interferon is necessary for the early control of proliferation evident in all strains but that the interferon response itself does not discriminate resistant from susceptible strains. The thymus dependency and radiation sensitivity of the early control of proliferation of Trypanosoma cruzi suggest that an immune mechanism(s) is involved in the early control of proliferation. The immune mechanism remains to be identified but it does not appear to require antibody (Trischmann 1983). It is our belief that further probing of the hostparasite interaction early after an infection will greatly enhance our understanding of the variable outcomes of the infection. ACKNOWLEDGMENTS I thank Stephen J. Davis for his excellent technical assistance. This work was supported by the U.S. National Institutes of Health Grant AI-20961. REFERENCES ALBRIGHT, J. W., AND ALBRIGHT, J. F. 1981. Differ-

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Znformaciones

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of Parasitology

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