key: cord-274247-5qiwui6u
authors: Torrecilhas, A C T; Faquim-Mauro, E; Da Silva, A V; Abrahamsohn, I A
title: Interference of natural mouse hepatitis virus infection with cytokine production and susceptibility to Trypanosoma cruzi
date: 1999-03-01
journal: Immunology
DOI: 10.1046/j.1365-2567.1999.00719.x
sha: 
doc_id: 274247
cord_uid: 5qiwui6u

Mouse hepatitis virus (MHV) infection can have a pronounced impact on several investigation areas. Reports on natural MHV outbreaks are rare and most studies have been conducted by deliberately infecting mice with MHV laboratory strains that cause moderate to severe disturbances to the immune system. We have investigated the effects of a natural acute outbreak of MHV in our otherwise specific-pathogen-free (SPF) inbred mouse colonies, and of enzootic chronic MHV infection on cytokine production and resistance to the intracellular pathogen Trypanosoma cruzi. We found that BALB/c and/or C57BL/6 SPF mice that had been injected with T. cruzi blood trypomastigotes from recently MHV-contaminated (MHV(+)) mice developed significantly higher parasite blood counts, accelerated death, and showed higher IL-10 production by spleen cells than their counterparts whose T. cruzi inoculum was derived from MHV-negative (MHV(−)) donors. Interferon-γ (IFN-γ) production by MHV(+) and MHV(−) mice was not significantly different. In contrast, T. cruzi infection of chronically MHV-infected mice did not result in major changes in the course of infection when compared with that observed in mice from MHV(−) colonies, although a trend to higher parasitaemia levels was observed in BALB/c MHV(+) mice. Nevertheless, both BALB/c and C57BL/6 T. cruzi-infected MHV(+) mice had diminished IFN-γ production to parasite-antigen stimulation in comparison with similarly infected MHV(−) mice. Interleukin-10 (IL-10) production levels by spleen cells did not differ between chronic MHV(+) and MHV(−) mice, but IFN-γ neutralization by monoclonal antibody treatment of anti-CD3-stimulated spleen cell cultures showed higher levels of IL-10 synthesis in MHV(+) BALB/c mice.

immune responses and on their interference with experimental models of infection. Most reports deal with virulent laboratory Mouse hepatitis virus (MHV ) collectively designates corona strains, although more recently, attenuated MHV laboratory viruses of a wide range of virulence. The strains that are strains have been used in an attempt to mimic the prevalent endemic in most mouse colonies over the world show relatively strains. 2 The objective of this study was to investigate the low virulence and animals from infected colonies do not effects of a natural acute outbreak of MHV due to accidental present overt signs of illness. Nevertheless, although tolerated exposure, in our otherwise specific-pathogen-free (SPF ) inbred by many researchers, evidence has accumulated over the years mouse colonies, and of enzootic chronic MHV infection on that results from several investigation areas can be comprocytokine production and resistance to the intracellular pathomised by concomitant MHV infections. In particular, the gen Trypanosoma cruzi. study of immunological parameters that determine resistance Trypanosoma cruzi is a dygenetic protozoan that infects to infections can be seriously affected by MHV infection several kinds of mammals and is the aetiological agent of (reviewed in refs 1,2) . There are few studies on the effects of Chagas' disease in man. 3 The parasite replicates in the cytoinfection by natural-low-virulence enzootic MHV strains on plasm of virtually any nucleated cell type including macrophages; non-dividing forms of the parasite are found free in more resistant.6 Susceptible mice have higher parasite numbers 3), pneumonia virus of mice (PVM ), minute virus of mice (MVM ), lymphocytic chorio-meningitis virus (LCMV ), in the blood and tissues and most animals die within 6 weeks of inoculation of as few as 50 parasites; in contrast, resistant Sendai virus, Ectromelia virus, mouse polio virus (GD7), and on ELISA for the bacteria Mycoplasma pulmonis. All pro-mice have lower parasite loads and survive infection with 50 000 parasites.7 Both T. cruzi-resistant and -susceptible cedures with the animals were in accordance with the principles of the 'Brazilian Code of Laboratory Animals Use'. mouse strains show elevated production of interferon-c (IFN-c) during infection8 and very low interleukin-2 (IL-2) and IL-4 production.8-11 IFN-c and tumour necrosis factor-a Trypanosoma cruzi infection, parasitaemia counting and experimental design (TNF-a), triggered by IL-12, are essential to control of parasitism by innate and acquired cellular immune systems in Infective blood trypomastigotes were obtained from Y strain T. cruzi-infected anaesthetized mice by drawing cardiac blood; mice.12-14 IL-10 exerts an important in vivo regulatory role on IFN-c and nitric oxide production12,15 and its absence in motile blood forms were counted and the desired number of parasites was injected intraperitoneally (i.p.). Infection was IL-10-gene-deprived mice12,16 or neutralization by monoclonal antibody (mAb) treatment17 results in lower parasitism, maintained by weekly i.p. infection of BALB/c mice. In the experiments designated 'acute' MHV infection, parasites were whereas increased parasitism occurs when mice are treated with recombinant IL-10 (rIL-10)12 or receive IL-10-and IL-maintained in mice coming from either recently infected MHV+ or from MHV− colonies and 500 or 5000 forms were 4-producing T cells.15 IL-4, depending on the parasite strain, is also involved in negative regulation of parasitism.18 inoculated in recipient BALB/c or C57BL/6 mice from MHV− colonies. In later experiments, designated 'chronic' MHV We found that BALB/c and C57BL/6 mice that had been injected with blood trypomastigotes from recently MHV-infection, the T. cruzi strain was started anew from tissueculture-grown trypomastigotes and maintained in MHV− contaminated mice became much more susceptible to T. cruzi infection and produced higher IL-10 levels than their counter-mice, whose blood was used as a source of T. cruzi to infect recipients derived from MHV+ colonies that had been infected parts whose T. cruzi inoculum was derived from MHVnegative (MHV−) donors. Comparison between mice coming for more than 4 months or from MHV− colonies. In these experiments the infective T. cruzi dose was 50, 500, or 5000 from chronically MHV-infected and from MHV− colonies, showed higher parasitaemia levels in BALB/c MHV-positive blood forms in BALB/c mice and 500, 5000 (not shown), 50 000, or 200 000 blood forms in C57BL/6 mice. As C57BL/6 (MHV+) mice but otherwise no major significant differences in susceptibility to T. cruzi. Nevertheless, quantitative differ-mice are much more resistant to T. cruzi infection, the high inocula would allow comparison between MHV− and MHV+ ences in IFN-c, IL-10 and nitric oxide production were found between MHV-infected and uninfected mice.

C57BL/6 colonies submitted to moderate to severe T. cruzi infection.7 Parasitaemia determination was performed by direct microscope (×40) counting of motile parasites in a 5 ml fresh MATERIALS AND METHODS blood sample, obtained from the lateral tail veins. Mice and MHV infection C57BL/6 and BALB/c female mice (8-10-week-old), acutely

Spleen cell cultures Spleen cell suspensions were prepared from T. cruzi-infected or chronically infected with MHV were obtained from Biotério de Camundongos Isogênicos do Departamento de Imunologia and uninfected mice, MHV+ or MHV−, depleted of erythrocytes by hypotonic lysis with distilled water and resuspended do ICB/USP (São Paulo, SP, Brazil ). Mice with negative serological tests for MHV, from both these strains, were in RPMI-1640 (Cultilab, Campinas, SP, Brazil ) complete medium containing 10% fetal calf serum (FCS; Cultilab) and obtained from Biotério de Camundongos Isogênicos da Universidade Estadual de Campinas (Campinas, SP, Brazil ).

supplemented with glutamine, 2-mercaptoethanol (2-ME ) and antibiotics as described.19 Spleen cell suspensions were pooled MHV− mice were housed and handled separate from and before those coming from MHV-contaminated animal facili-from three mice and were cultured in duplicate or triplicate in 24-well flat-bottomed plates at 107/ml or at 5×106/well ties. The mice were fed autoclaved food and water, and were handled using disposable gloves. MHV infection was diagnosed and stimulated with T-Ag (5×106 Frozen-Thawed tissue culture trypomastigotes parasites) prepared as described.9 by antibody testing of the sera. The MHV enzyme-linked immunosorbent assay ( ELISA) diagnostic kits sold by Charles Concanavalin A (Con A at 2·5 m/ml ) or plate-bound anti-CD3 [mAb 145-2C11, Americal Type Culture Collection (ATCC ) River Laboratories ( Wilmington, MA) were used according to the manufacturer's instructions. When the outbreak of CRL 1973], coated at 10 mg/ml, 500 ml/well ) were also used as T-cell stimulants.11. Supernatants from the cultures were MHV was detected, levels of anti-MHV antibodies were very high with corrected optical density (OD) values for sera harvested after 20 hr from the higher cell-density cultures and after 72 hr from the lower-density cultures. The following ranging from 8·5 to 23·8 (positive test values >3). These were calculated (as indicated by the manufacturer) by the following neutralizing rat mAb anti-mouse cytokines were incorporated to the cultures in some experiments: JES5-2A5 anti-IL-10, and formula: [(OD obtained for the test serum diluted at 1/50 incubated with the cells containing the virus) -(OD obtained XMG1.2 anti-IFN-c; both were used at final concentrations of 20 mg/ml. The anti-CD4 mAb GK1.5 was added to cultures for the same serum and dilution incubated with uninfected cells)]/0·13. Fluorescence antibody testing was also performed at 10 mg/ml. as an additional control and ranked + + + + for serum incubated with MHV-infected cells. The colonies were serologi-Cytokine assays Cytokine levels in the culture supernatants were measured by cally negative on ELISA and immunofluorescence testing for the following viruses: respiratory-enteric orphan virus (Reo two-site sandwich ELISA using the following mAb pairs of which the second cited was biotinylated. IFN-c, XMG1.2 and when the inoculum came from MHV− donor mice. The AN18; IL-10, JES-2A5 and SXC-1; IL-4, 11 B 11 and differences in parasitaemia could be observed with inocula of BVD6 24G2.11 Minimum levels of detection for the assays 500 or 5000 blood trypomastigotes (Fig. 1a,b) . Moreover, for were:. IFN-c, 1·56 ng/ml; IL-10, 3·25 units/ml; and IL-4, BALB/c mice injected with MHV+ inocula, mortality reached 0·156 ng/ml. The rat anti-mouse cytokines producing 80% by day 20 (500 T. cruzi forms) and 100% by day 16 hybridomas were a generous gift from Dr R. L. Coffman, (5000 T. cruzi forms) whereas all recipients of MHV− inocula DNAX Research Institute of Molecular and Cellular Biology, survived to 30 days after infection. All C57BL/6 mice Palo Alto, CA. Standard curves were obtained with recombisurvived after infection whether inoculated with T. cruzi from nant mouse cytokines. The supernatants were tested in serial MHV+ or MHV− donor mice. twofold dilutions and the results were expressed as the arith-Serological tests for MHV became positive in T. cruzimetic mean of duplicate determinations. The SD did not infected (from MHV+ donors) C57BL/6 mice by day 28 after exceed 20% of the mean.

infection and in 30% of a group of 20 BALB/c mice that survived to 28 days after being infected with 50 T. cruzi Statistical analysis forms. Recipients of inocula from MHV− mice remained The significance of differences in parasitaemia between distinct serologically negative for MHV. Looking into possible causes experimental groups was examined by analysis of variance with of the observed increase in susceptibility to T. cruzi of mice repeated measurements followed by Tukey's test for multiple infected with MHV+-derived inocula. we investigated whether comparisons. Differences of 0·5 log 10 or over were significant cytokine production was altered concomitant to the viral at least at P<0·05. Differences in cytokine production levels infection. Spleen cells (stimulated with Con A or T-Ag) from were tested by Bonferroni's multiple comparison test.

mice that received an MHV+ inoculum of 500 T. cruzi produced detectable and much higher levels of IL-10 during the first 3 weeks of infection than spleen cells from mice that RESULTS

received an identical inoculum of T. cruzi derived from T. cruzi infection and cytokine production in MHV− mice MHV− mice. In fact, IL-10 production in this last situation inoculated with T. cruzi blood forms obtained from donor-mice was very low and often below 3·12 units/ml (Table 1 ). IL-10 that were serologically MHV+ or MHV− production, on day 11 after infection, in BALB/c (but not in C57BL/6) mice was CD4-activation dependent as treatment Our preliminary observation, suggestive of an infection with GK1.5 mAb suppressed most of its synthesis outbreak in the mouse colonies, was increased susceptibility (63 units/ml in untreated versus 6 units/ml in GK1.5 treated to T. cruzi infection first detected in BALB/c mice. As the cultures in Con A-stimulated and 22 units/ml versus diagnosis of MHV infection was confirmed, we first investi-6 units/ml, respectively, in T-Ag-stimulated BALB/c spleen gated how a T. cruzi inoculum derived from donor mice that cell cultures). In C57BL/6 mice, the values were 14 units/ml had concomitant MHV infection would compare with a in untreated versus 10 units/ml in GK1.5-treated Con similar inoculum originated in MHV− mice in regard to their A-stimulated cultures and 13 versus 10 units/ml, respectively, course of infection in a T. cruzi-susceptible (BALB/c) and in after T-Ag stimulation. In spite of the higher IL-10 proa resistant (C57BL/6) mouse strain. As shown in Fig. 1 , duction by spleen cells from mice that had received the parasitaemia levels during the first 9 days of infection were MHV+ inoculum, IFN-c levels were not significantly different significantly higher for both strains of mice when infected with T. cruzi blood forms derived from MHV+ donors than from those secreted by spleen cells from MHV− T. cruzi recipients and IL-4 production was below detection levels in resulted in significant increases in IFN-c production of the order of 40-100% (Fig. 2) . We next investigated whether all groups (data not shown).

infecting MHV− or mice that were chronically infected MHV with T. cruzi would affect cytokine production and/or alter Cytokine production by spleen cells from mice derived from the course of infection.

Spleen cell cultures from BALB/c and C57BL/6 mice coming

In contrast with the marked increase in parasitaemia and (data not shown). Production of IFN-c by spleen cells from susceptibility to T. cruzi determined by the situation of acute MHV+ C57BL/6 and BALB/c mice cells to Con A stimulation simultaneous infection with blood parasites derived from was not significantly higher than in spleen cell cultures from acutely infected MHV+ donors as described above, no statis-MHV− animals (Fig. 2) . Production of IFN-c to Con A was tically significant differences of parasitaemia levels or mortunder control by IL-10 both in MHV− and in MHV+ mice ality were observed between chronically infected MHV+ and because the addition of anti-IL-10 mAb 2A5 to cultures MHV− BALB/c or C57BL/6 mice infected with T. cruzi. However, peak T. cruzi parasitaemia levels were attained earlier and were 30-50% higher in BALB/c MHV+ mice as compared to MHV-free mice (data not shown). Differences in cytokine production between T. cruziinfected MHV+ and MHV-free groups of mice were only observed in the first week of infection. BALB/c mice spleen cell cultures from MHV+ mice infected with T. cruzi produced lower IFN-c levels to Con A and T-Ag stimulation than cultures from MHV− mice (Fig. 3) . Although the potential to produce IFN-c to polyclonal Con A stimulation was preserved in MHV+C57BL/6 mice, IFN-c production was markedly suppressed on parasite-specific stimulation (Fig. 4) . The comparison of IL-10 production levels among BALB/c and C57BL/6 mice, MHV− or MHV+, infected with T. cruzi yielded no significant differences (data not shown). Nevertheless, when BALB/c (but not C57BL/6) spleen cell uninfected MHV+ mice (Fig. 5, normal group) , suggesting mice, suppression of IFN-c responses is limited to the antigenspecific compartment whereas MHV infection affects more that the potential to increased IL-10 production stimulated by MHV infection was, in these mice, maintained in check by severely BALB/c mice by suppression of both polyclonal and specific IFN-c responses. IFN-c. Taken together, these results indicate that in C57BL/6

immune response have been performed in situations of deliberate infection of mice with MHV laboratory strains of varying Enzootic MHV strains are of low virulence, mostly entervirulence that cause moderate to severe disturbances to the otropic and notoriously difficult to isolate and to grow in immune system. Mice infected with the pantropic strain of vitro.1 When the outbreak was detected in the mouse colony, medium-virulence, JHM, show suppression of Con A-induced we unsuccessfully tried to isolate infective viral particles from proliferation, decreased IL-2 and IL-4 synthesis and a delay the plasma and liver of seropositive mice. Immunosuppressing in IFN-c production in the first week of infection, whereas, the animals with cyclophosphamide also failed to promote later in infection, large amounts of IFN-c are produced by viral isolation. The difficulty in isolating low virulence enzootic BALB/c mice.25-27 Macrophage function was impaired in mice enterotropic MHV, as opposed to polytropic laboratory MHV infected with this strain28 or with naturally occurring MHV strains, has been frequently reported.1,2,20 We had (as many strains.29,30 Increased message levels for IFN-c, IL-4, IL-10, other authors) to rely on serum antibody screening and TNF-a and inducible nitric-oxide synthase (iNOS ) were found serological conversion as a criterion to identify occurrence of in the brain of mice infected with a neurotropic JHM variant MHV infection. Serological testing, although highly specific, strain.31 MHV A-59 is yet another laboratory strain, but does not distinguish between acute and chronic MHV infection of relatively low virulence that, although subclinical, is and thus we will discuss the immunological alterations found accompanied by increased production of IFN-c and suppresin the group of mice that had recently become seropositive for sion of Con A spleen responses.32,33 MHV (recent MHV-testers) in comparison with those found

There have been few reports on immunological alterations in mice originating from colonies with longstanding record of resulting from natural MHV outbreaks. However, established MHV seropositivity. Both groups of mice were serologically 'chronic' natural infection with MHV was reported to affect negative on testing for several other mouse pathogenic viruses mostly splenic T lymphocytes, with a 20-50% decrease in and for Mycoplasma pulmonis (see the Materials and Methods).

proliferative responses to Con A and resistance to the effects We have found a much more severe course of T. cruzi of nor-adrenalin or dibutyryl-cAMP. 34 We found that lymphoinfection in MHV− mice infected with blood forms obtained proliferative responses of spleen cells from BALB/c and from mouse colonies that had recently become seropositive C57BL/6 mice, MHV+ or MHV− recipient mice to Con A, for MHV, as compared with mice inoculated with parasites T-Ag, or anti-CD3 stimulation were not significantly different. derived from MHV− donors; increased susceptibility to infec-Suppressed lymphoproliferative responses to these stimuli, tion was accompanied by increased synthesis of IL-10 by commonly seen in the course of T. cruzi infection,11 were spleen cell cultures. Interleukin-10 down-regulates IL-12 and observed from day 11 of infection, with minimal levels of IFN-c production, besides antagonizing IFN-c-and TNF-asuppression seen on day 19 and recovery by day 27 (data not dependent macrophage activation and intracellular T. cruzi shown). Thus, in the course of T. cruzi infection, we did not killing.12,13,17,21-23 In spite of the enhanced IL-10 secretion by observe, neither in recently seropositive recipient mice nor in spleen cells from MHV+ recipients, no decrease of IFN-c mice from chronically MHV-infected colonies, suppression of levels, in comparison to recipients of MHV− donors could be lymphoproliferation to Con A, to parasite antigens or to antidetected in these same cultures. However, the data on enhanced CD3 stimulation that could be ascribed to the viral infection. IFN-c synthesis by anti-IL-10 mAb treatment, showed that However, its occurrence could have been masked by the intense endogenous IL-10 was down-regulating IFN-c production in suppression characteristic of acute T. cruzi infection. this situation of probable acute concomitant infection with Our results on the aggravation of T. cruzi infection in the parasite and the virus. The maintenance of in vitro IFN-c BALB/c and C57BL/6 mice by using parasite inocula mainsecretion rates in the presence of increased IL-10 concentained in mice that had positive serology for MHV are in trations has been described upon treatment of T. cruzi-infected agreement with a previous report on CBA/J mice infected with mice with high doses of rIL-10 that aggravate infection.12

T. cruzi derived from corona virus-positive mice.35,36 These Both mouse strains, BALB/c and C57BL/6, respectively, authors were able to demonstrate, in the plasma, infective susceptible and resistant to T. cruzi, showed increased parasitavirus particles that could be neutralized by anti-MHV antiemia and augmented IL-10 production, but increased mortality serum. Nevertheless, the underlying mechanisms leading to as a consequence of MHV infection was not observed for increased susceptibility to the parasite were not explored. C57BL/6 mice and they maintained their resistant phenotype

We further investigated the influence of a longstanding to T. cruzi infection. Although mouse strain resistance to

(1 year) established endemic MHV infection on the course of MHV infection is highly dependent on the MHV strain, studies T. cruzi infection and immune response. In contrast with the with laboratory MHV-strains have concluded that indifferently marked effects on the immune response observed in MHV− to the degree of MHV-strain virulence, replication in macrohosts infected with T. cruzi derived from recently MHV+ mice, phages does always occur.24 Viral replication inside macrothe disturbances of immune response found in mice from phages may directly interfere with T. cruzi killing ability by chronically MHV-infected colonies were milder. Production these cells and stimulate a number of macrophage functions of IL-10 was similar between MHV+ and MHV− mice, while including IL-10 synthesis. In this regard, C57BL/6 mice are parasite-antigen-stimulated IFN-c production was lower in T. semisusceptible to most MHV strains;1,2 as production of cruzi-infected BALB/c and C57BL/6 mice. However, when IL-10 by MHV-infected C57BL/6 mice (but not by BALB/c neutralization of IFN-c in the cultures unmasked the full mice) was CD4+-activation independent, virus-infected or potential of IL-10 secretion, MHV+ BALB/c mice (but not virus-stimulated macrophages could be the main source of C57BL/6), showed higher IL-10 production levels. This result this cytokine.

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