Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites

Abstract

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαβ+CD8+IFN-γ+ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαβ+CD4+ T cells were also found to increase in the infected mice, however later than CD8+ T cells. Interestingly, splenic and MLN CD4+CD25+ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαβ+CD8+ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.

Figure 1

cDC and pDC produce IL-12 in response to N. caninum i.g. infection. (a) Representative example of flow cytometric analysis of surface PDCA-1 and CD11c expression on total MLN leukocyte cells. Dot plots represent cells collected from C57BL/6 mice 18 h after i.g. challenge with 5 × 10⁷N. caninum tachyzoites. Gates were set as shown to delineate cDC (CD11c^high) and pDC (CD11c^low PDCA-1^high). (b) Proportion of IL-12⁺ cells within cDC and pDC populations in the MLN of C57BL/6 mice, evaluated by flow cytometric analysis, at the specified time points after i.g. treatment with PBS (open bars) or i.g. inoculation with 5 × 10⁷ N. caninum tachyzoites (closed bars). Bars represent the mean plus one standard deviation of three animals in the PBS group and four animals in the infected mice group. This is one representative result of two independent experiments (*P<0.05; **P<0.01).

Figure 2

Increased expression of IFN-γ by TCRβ⁺CD8⁺ IEL in N. caninum infected mice. Representative dot plots of (a) cells isolated from the small intestines of C57BL/6 mice (IEL were gated as shown); (b) TCRγδ and TCRβ IEL. (c) Gated TCRβ CD8⁺ IEL expressing IFN-γ in control (PBS) and N. caninum-infected mice (NcT). (d) Frequency of TCRβ⁺CD8⁺ IFN-γ⁺ IEL in control and N. caninum i.g.-infected mice, 48 h after challenge. Bars represent mean plus one standard deviation of three animals in the PBS group and four animals in the infected mice group. This is one representative result of three independent experiments (*P<0.05).

Figure 3

Expression of IFN-γ in splenic and MLN CD8⁺ and CD4⁺ T cells. Scatter plots of frequency of CD8⁺IFN-γ⁺ T cells (a and b) or CD4⁺IFN-γ⁺ T cells (c and d), as indicated, in the MNL (a and c) and spleen (b and d) of control (PBS) or N. caninum-infected mice (NcT), 4 and 7 days upon i.g. challenge. Each symbol represents an individual mouse; horizontal bars correspond to mean values of the respective group. Five mice per group were used. This is one representative result of three independent experiments. Statistical significance between groups is indicated above symbols (*P<0.05; **P<0.01).

Figure 4

Spleen and MLN Treg and Teff cell numbers. Scatter plots of CD4⁺CD25⁺ Treg (Foxp3⁺) and Teff (Foxp3^-) cell numbers in the MLN and spleen of control (open squares) or N. caninum-infected mice (closed triangles), 4 and 7 days upon the i.g. challenge. Each symbol represents an individual mouse; horizontal bars correspond to mean values of the respective group. Five mice per group were used. This is one representative result of three independent experiments. Statistical significance between groups is indicated above symbols (*P<0.05; **P<0.01).

Figure 5

High suppressive activity of Treg from N. caninum-infected mice. (a) Flow cytometric evaluation of anti-CD3 mAb (1 μg/mL) induced proliferative response of 2.5 × 10⁴ CFSE-labelled naïve CD4⁺CD25^- T (responder) cells cultured for 72 h with 10⁵ irradiated APC/well, in the absence (Pos. control) or presence of CD4⁺CD25⁺ T cells obtained from control mice or mice infected with N. caninum. Neg. control corresponds to responder cells with no mAb added. Histograms correspond to 1:1, 2:1 and 10:1 responder: CD4⁺CD25⁺ cell ratios or 1:1 responder: CD4⁺CD25^- T cell ratio, as indicated. (b) Flow cytometric evaluation of N. caninum antigen-induced proliferative response of 2.5 × 10⁴ CFSE-labelled splenic CD4⁺CD25^- T (responder) cells obtained from 7 day-infected mice, cultured for 72 h with 10⁵ antigen-loaded BMDC/well, in the absence (Pos. control) or presence of CD4⁺CD25⁺ T cells obtained from control mice or mice infected with N. caninum. Non-specific proliferation control (Neg. control) corresponds to responder cells cultured with LPS (50 ng/mL) activated BMDC, without N. caninum antigen. Histograms correspond to 1:1 and 2:1 responder: CD4⁺CD25⁺ cell ratios, as indicated. Numbers within histograms correspond to the percentages of cells that divided at least once. CD4⁺CD25⁺ cells added in each condition were sorted from pooled splenic cells of 5 mice per group. Results are a representative example out of three (a) or one out of two (b) independent experiments.

Figure 6

Suppression of cytokine production by splenic and MLN Treg. IFN-γ and IL-10 cytokine concentration in the supernatants of MLN or splenic naïve 2.5 × 10⁴ CD4⁺CD25^- T cells cultured for 72 h with anti-CD3 mAb (1 μg/mL) and 10⁵ APC, alone or in the presence of 1:1 CD4⁺CD25⁺ MLN or splenic T cells from control or N. caninum-infected mice, as indicated. Each symbol represents an individual culture well. Horizontal bars represent the mean values of the respective group. CD4⁺CD25⁺ cells added in each condition were sorted from pooled MLN or splenic cells of 5 mice per group, as indicated. Results are a representative example out of three independent experiments. Statistical significance among groups was determined by one-way ANOVA, followed by a Bonferroni post-test, and is indicated above symbols (**P<0.01; ***P<0.001).