Characterization of the B-cell immune response elicited in BALB/c mice challenged with Neospora caninum tachyzoites

Abstract

Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite-evasion immune-mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69-expressing peritoneal and splenic B cells. This early B-cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin-secreting splenic B cells and a rise in serum levels of N. caninum-specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum-infected mice. The B-cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with gamma-irradiated parasites. Contrasting with the peripheral B-cell expansion, a depletion of B-lineage cells was observed in the bone-marrow of the N. caninum-infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite-specific antibodies nevertheless impaired interferon-gamma (IFN-gamma) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non-lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti-N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite-specific, non-polyclonal, B-cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN-gamma in the host protective immune mechanisms against this parasite.

Figure 8

Increased susceptibility to neosporosis in mice immunized with N. caninum antigens. Survival rates of mice infected i.p. with 5 × 10⁶ NcT 30 days after the second i.d. inoculation at a 3-week interval of (a) alum (open circles) or 10 µg of NcS in alum adjuvant (closed triangles), (b) Freund's adjuvant (open circles) or 10 µg of NcS in Freund's adjuvant (closed lozenges), (c) PBS (open circles) or 5 × 10⁵ gamma-irradiated NcT in PBS (closed squares). The survival rate of mice sham-immunized was significantly different from that of immunized mice immunized in all the groups (median survival time in days (MST) = 7 (95% CI: 6; 8) chi-square = 12·1, d.f. = 1, P < 0·0006; MST = 8 (95% CI: 7; 9) chi-square = 11·4, d.f. = 1, P < 0·0008; MST = 9 (95% CI: 8; 10) chi-square = 8·1, d.f. = 1, P < 0·005, for results shown in panels A, B and C, respectively). Six mice per group were used. This is one representative result of two independent experiments.

Figure 1

Early B-cell activation induced in vitro and in vivo by N. caninum. (a) Expression of CD69 on the surface of BALB/c mice splenic IgM⁺ cells as evaluated by flow cytometric analysis 14 hr after in vitro stimulation with medium alone (None), with 5 µg/ml of LPS (LPS) as a positive control or with 10²−10⁵N. caninum tachyzoites/ml (NcT) as indicated. Bars represent means plus 1 SD of triplicated well samples for each indicated group. This is one representative result of three independent experiments. (b and c) Numbers of BALB/c mice peritoneal (b) or splenic (c) CD69⁺ IgM⁺ cells, 14 hr after i.p. treatment with PBS (Control), 12·5 µg of LPS (LPS) or 5 × 10⁵ NcT. Bars represent the mean plus one SD of three mice per control groups and four mice per N. caninum-treated groups. This is one representative result of three independent experiments. In this and in the following figures statistically significant differences between control and N. caninum-stimulated groups were indicated (*P < 0·05; **P < 0·01).

Figure 2

B-cell population kinetics in the spleen. Numbers of IgM⁺ CD5^– (a) and of IgM⁺ CD5⁺ (b) B cells in the spleen of BALB/c mice evaluated by flow cytometric analysis on the indicated days after i.p. injection with PBS (open bars), 5 × 10⁵N. caninum tachyzoites (closed bars) or 5 × 10⁵N. caninumγ-irradiated tachyzoites (dashed bars). Bars represent the mean plus 1 SD of three mice per control groups and four mice per N. caninum-treated groups. This is one representative result of three independent experiments. ND, not done.

Figure 3

Effect of N. caninum challenge on B-cell maturation in the bone-marrow. Quantification of B-cell precursors (a) in the bone marrow of BALB/c mice 3 days after injection i.p. with PBS or with 5 × 10⁵N. caninum tachyzoites (NcT). Figures shown in the lower dot-plots above the respective regions represent numbers in millions of B220⁺ IgM^– and B220⁺ IgM⁺ bone-marrow cells displaying lymphoid forward and side scatter parameters (gated as shown in the upper dot-plots) observed in PBS- or NcT-infected mice as indicated. Numbers represent the mean ± one SD of three mice in the control group and four mice on the N. caninum-infected group. This is one representative result of three independent experiments. Quantification of apoptotic B-lineage (B220⁺) cells (b) gated as described above, observed in the bone marrow of BALB/c mice 24 hr after PBS- or NcT-inoculation as indicated. Regions on the figure identify early apoptotic (annexin V⁺) B220⁺ cells (Early) and late apoptotic/necrotic (propidium iodide⁺) cells (Late). Represented is one typical result from each mice group of an experiment repeated three times. The mean numbers ± 1 SD of early apoptotic cells were of 92 × 10³ ± 3·5 × 10³ and 234 × 10³ ± 76 × 10³ for PBS- or NcT-inoculated mice, respectively (n = 4 on each group, P < 0·05). No significant difference in the numbers of late apoptotic/necrotic cells between these two mice groups was observed.

Figure 4

Increased serum antibody titres in NcT-infected mice. Serum ELISA titres of total (a) or NcS-specific (b) antibodies of the indicated isotypes detected in mice sera collected 15 days after i.p. inoculation with PBS (open bars), 5 × 10⁵N. caninum tachyzoites (closed bars) or 5 × 10⁵N. caninumγ-irradiated tachyzoites (dashed bars). Bars represent the mean plus 1 SD of four mice per control groups and six mice per N. caninum-infected groups. This is one representative result of three independent experiments. ND, not detected.

Figure 5

Increased IFN-γ production in BALB/c mice challenged with N. caninum tachyzoites. Levels of IFN-γ mRNA expression, normalized to HPRT mRNA (a) detected by real time RT–PCR in the spleen of BALB/c mice inoculated i.p. with PBS (controls, open bars) or 5 × 10⁵N. caninum tachyzoites (closed bars), 7 and 15 days after challenge. Bars represent the mean plus 1 SD of four mice per group. This is one representative result of two independent experiments. Serum ELISA titres of IFN-γ (b) detected in mice sera collected 7 and 15 days after i.p. inoculation with PBS (open bars) or 5 × 10⁵N. caninum tachyzoites (closed bars). Bars represent the mean plus 1 SD of four mice per group. This is one representative result of three independent experiments. ND, not detected.

Figure 6

Expression of costimulatory molecules on B cells is increased in NcT-infected mice. Flow cytometric analysis of PNA binding and of CD80 or CD86 expression on the surface of splenic cells from BALB/c mice 15 days after i.p. inoculation with PBS (PBS), 5 × 10⁵γ-irradiated NcT (Irr-NcT) or with 5 × 10⁵ NcT (NcT). Figures shown inside the dot plots are means ± 1 SD of the numbers (in millions) of PNA⁺, CD80⁺ or CD86⁺ B cells (B220⁺ cells) as indicated, on each mouse group. Four mice per group were used and the results are representative of one experiment repeated three times. Also shown on the far right is a representative example of MHC class II expression observed on the surface of PNA^low (grey histogram) and PNA^high (overlay open histogram) B220⁺ cells in NcT-infected mice.

Figure 7

Anti-NcS antibody titres in NcS- or irradiated-NcT immunized BALB/c mice. Immunoglobulin ELISA titres of anti-NcS antibodies of the indicated isotypes detected in mice sera 30 days after the second i.d. inoculation with the different immunogenic preparations (right closed bars on each panel), from left to right, 10 µg of NcS in alum adjuvant, 10 µg of NcS in Freund's adjuvant, 5 × 10⁵γ-irradiated NcT or with control preparations (left open bars): alum adjuvant, Freund's adjuvant, PBS, respectively. Bars represent the mean plus 1 SD of six mice per group. This is one representative result of two independent experiments. ND not detected.

Figure 9

Decreased IFN-γ mRNA expression, comparatively to sham-immunized controls, in BALB/c mice immunized with N. caninum antigens and challenged with N. caninum tachyzoites. Levels of IFN-γ mRNA expression, normalized to HPRT mRNA, detected by real time RT–PCR, 6 days after challenge i.p. with 5 × 10⁶N. caninum tachyzoites, in the spleen of mice (a) sham-immunized with alum (open bars) or immunized with NcS in alum adjuvant (closed bars); (b) sham-immunized with Freund's adjuvant (open bars) or immunized with NcS in Freund's adjuvant (closed bars); (c) sham-immunized with PBS (open bars) or immunized with irradiated NcT in PBS (closed bars). Bars represent the mean plus 1 SD of three mice per group. This is one representative result of two independent experiments.