B cells modulate T cells so as to favour T helper type 1 and CD8+ T-cell responses in the acute phase of Trypanosoma cruzi infection

Abstract

In this study, we have evaluated the production of pro- and anti-inflammatory cytokines and the formation of central and effector memory T cells in mice lacking mature B cells (mu MT KO). The results show that Trypanosoma cruzi infection in C57Bl/6m mu MT KO mice is intensified in relation to control mice and this exacerbation is related to low levels of inflammatory cytokines produced during the acute infection and the lower numbers of central and effector memory CD4(+) and CD8(+) T cells generated during the acute phase of the infection. In addition, a marked reduction in the CD8(+) T-cell subpopulation was observed in mu MT KO infected mice. In agreement to this, the degree of tissue parasitism was increased in mu MT mice and the tissue inflammatory response was much less intense in the acute phase of the infection, consistent with a deficit in the generation of effector T cells. Flow cytometry analysis of the skeletal muscle inflammatory infiltrate showed a predominance of CD8(+) CD45Rb low in B-cell-sufficient C57Bl/6 mice, whereas the preponderant cell type in mu MT KO skeletal muscle inflammatory infiltrate was CD4(+) T cells. In addition, CD8(+) T cells found in skeletal muscle from mu MT KO infected mice were less activated than in control B-cell sufficient infected mice. These results suggest that B cells may participate in the generation of effector/memory T cells. In addition and more importantly, B cells were crucial in the maintenance of central and effector memory CD8(+) T cell, as well as the determination of the T cell cytokine functional pattern, and they may therefore account for critical aspects of the resistance to intracellular pathogens, such as T. cruzi.

Figure 1

B cells help to control parasitaemia. In (a) and (b) the levels of parasitaemia were determined on the indicated days after an initial infection with 5 × 10² or 5 × 10³ trypomastigote forms, respectively (C57Bl/6 mice, closed squares; C57Bl/6 muMT KO mice, closed circles). Each point represents the mean of the parasitaemia values from mice of the different groups. (c, d) Shows the rate of mortality in the different experimental groups: C57Bl/6 mice (filled bars) and C57Bl/6 muMT KO mice (hatched bars), n = 10 animals/group in the beginning of the experiment. The results shown in (a) and (b) were compared in each point using the Mann–Whitney test (*P < 0·05, **P < 0·01). The mortality rates in (c, d) were compared, using Mann–Whitney U-test.

Figure 2

Spleen cells from T. cruzi-infected C57Bl/6 muMT KO mice produce less inflammatory cytokines (IFN-γ, IL-12), and similar or increased amounts of IL-18, in comparison with control mice. Spleen cells from control or infected C57Bl/6 or C57Bl/6 muMT KO mice were cultured in vitro in the presence of medium alone. Three to four spleens from each group of mice were pooled and used to prepare the cell suspensions. (a, b, c) The levels of IFN-γ (ng/ml), IL-12 (ng/ml) or IL-18 (ng/ml) production by spleen cells from the different experimental groups after 48 hr of culture. (d, e, f) The amounts of IFN-γ (ng/ml), IL-12 (ng/ml) or IL-18 (pg/ml) in the sera from the different experimental groups (day 30 after infection). Cytokines were measured by ELISA, as described in the Methods section. Columns represent the mean of the amount of cytokines of triplicate cultures or three to four different serum samples. The data are from one of three experiments with similar results. Mann–Whitney test was used. (*P < 0·05, **P < 0·01; ND, not detected)

Figure 3

Spleen cells from B-cell deficient mice produce higher amounts of IL-4 and similar levels of IL-10 in comparison with control mice during the acute phase of T. cruzi infection. Spleen cells from control or infected C57Bl/6 or C57Bl/6 muMT KO mice were cultured in vitro in the presence of medium alone. Three to four spleens from each group of mice were pooled and used to prepare the cell suspensions. (a and b) The levels of IL-4 (pg/ml) and IL-10 (pg/ml) production by spleen cells from the different experimental groups after 48 hr of culture (day 30th after infection). Cytokines were measured by ELISA, as described in Methods. Columns represent the mean of the amount of cytokines of triplicate cultures. The data are from one of three similar experiments. Mann–Whitney test, *P < 0·05.

Figure 4

T. cruzi-infected B-cell deficient mice have reduced numbers of CD8⁺ splenic T cells and impaired generation of central or effector splenic memory T cells. Spleen cells from control or infected C57Bl/6 and C57Bl/6 muMT KO mice, were counted, stained and evaluated by FACS as described in Methods. Frequencies of CD44 high × CD62L high T cells (central memory T cells) and CD44 high × CD62L low T cells (effector memory T cells) are shown in the upper and lower density plots inside electronically gated CD4⁺ or CD8⁺ T cells, respectively. Numbers inside plots represent the relative frequency of each T-cell subpopulations. Density plots are representative of one experiment where spleen cells were pooled for each experimental group (three to four spleens/group). Other two independent experiments were done where stainings were performed in individual mouse and the data was used to calculate the total number of CD4⁺ and CD8⁺ T cells showed in the bar chart on the right. The columns represent the mean number of each splenic T-cell subpopulation (n = 4–7 mice/group). Lower dot plots represent one experiment where the frequencies of splenic central and memory CD4⁺ (left) or CD8⁺ (right) T cells were evaluated in individual mouse. Vertical bars represent the standard deviation of the mean. Student's t-test was used to compare the indicated groups. Mice were infected with 5 × 10² parasites and were analysed 30 days after infection (*P ≤ 0·05, **P ≤ 0·01, ***P ≤ 0·005).

Figure 5

B-cell deficient mice have smaller percentages of activated/memory CD8⁺ T cells in inflammatory infiltrates. Skeletal muscle mononuclear cells (SMMC) from infected C57Bl/6 and C57Bl/6 muMT KO mice were separated, stained and evaluated by flow cytometry as described in the Methods section. Upper density plots show the percentage of SMMC CD4⁺ and CD8⁺ T cells in the respective experimental groups, as indicated. Frequencies of CD45Rb low and high T cells are shown in the lower histograms inside electronically gated CD4⁺ or CD8⁺ T cells, respectively. Numbers inside plots and histograms represent the relative frequency of each T-cell subpopulations. Histograms are representative of one out of three experiments with similar results. Pooled SMMC were obtained from three to four mice/group.

Figure 6

B-cell deficient mice have decreased inflammatory infiltrate and augmented tissue parasitism during the acute phase of T. cruzi infection. Wild-type C57Bl/6 or muMT KO C57Bl/6 mice were infected i.p. with 5 × 10² trypomastigote forms of Tulahuem strain of T. cruzi. Quantitative analysis of inflammatory infiltrates (a) and the numbers of intact T. cruzi parasite nests (b) in skeletal muscle tissues were evaluated in 10 non-consecutive histological sections obtained from mice at day 20 after infection, as described in Materials and Methods. Results are shown as mean ± SD. Student's t-test was used to compare indicated groups. Two and three asterisks indicate that differences are statistically significant (P ≤ 0·01 and P≤0·005, respectively) between the indicated groups (n = 8–10 mice/group). Representative skeletal muscle sections from wild-type C57Bl/6 and muMT KO C57Bl/6 infected mice (c and d, respectively) (magnification × 100) are shown. Similar results were obtained in four independent experiments.