Induction of cell-mediated immunity to Staphylococcus aureus in the mouse mammary gland by local immunization with a live attenuated mutant

Abstract

The efficacy of intramammary (Ima) immunization with a live attenuated (la) Staphylococcus aureus mutant to protect the mouse mammary gland from infection has previously been established. The present study was aimed at evaluating whether Ima immunization with la-S. aureus can induce cell-mediated immune responses to the pathogen within the mammary gland. Mice were immunized by Ima route with la-S. aureus, and regional lymph node mononuclear cells were obtained thereafter. A higher expression of the interleukin-2 receptor was found on B and T cells from immunized mice when they were compared with control mice. Immunization with la-S. aureus induced strong proliferative responses to S. aureus. Moreover, significantly increased levels of gamma interferon (IFN-gamma) were produced by CD4+ T cells when lymphocytes from immunized mice, but not from control mice, were cultured in the presence of staphylococcal antigens. Moreover, a significant increase in the percentage of IFN-gamma-producing CD4+ and CD8+ T cells was observed after S. aureus Ima challenge in immunized mice compared to challenged control mice. Our results demonstrated that Ima immunization with la-S. aureus induced primed lymphocyte populations capable of responding against staphylococcal antigens during in vitro stimulation, as well as during in vivo infection by S. aureus. CD4+ and CD8+ T cells appear to be the main lymphocyte subpopulations involved in this response. It is suggested that IFN-gamma production induced by Ima immunization may play a pivotal role in the eradication of intracellular staphylococci.

FIG. 1.

In vitro proliferative responses of mammary gland lymph node cells to S. aureus. Cells from regional mammary gland lymph nodes of control (left panels) and immunized mice (right panels) were cultured in the presence or absence of whole heat-killed S. aureus (A) or S. aureus total proteins (B). Lines connect the counts per minute (cpm) measured in the presence or absence of antigen at the individual level. Horizontal short lines indicate the median cpm value obtained for each group. P values were obtained by using the Wilcoxon signed-rank test for paired samples by comparing the median cpm measured after stimulation with S. aureus or staphylococcal proteins versus the median cpm measured in medium (✽, P < 0.01; ✽✽, P < 0.001). One representative experiment of three is shown.

FIG. 2.

Effect of Ima immunization on cytokine production in response to S. aureus. Mononuclear cells from mammary gland lymph nodes of control mice (left panels) and immunized mice (right panels) were cultured in the presence of medium alone, heat-killed wt-S. aureus (A), total S. aureus proteins (B), or heat-killed S. aureus of bovine origin (strain 319) (C). Lines connect IFN-γ levels measured in the presence or absence of antigen at the individual level. Horizontal short lines indicate the median values obtained for each group. P values were determined by using the Wilcoxon signed-rank test for paired samples in order to compare median IFN-γ levels measured after stimulation with S. aureus or staphylococcal proteins versus the median IFN-γ levels measured in medium (✽, P < 0.01; ✽✽, P < 0.001). One representative experiment of three is shown.

FIG. 3.

Intracellular IFN-γ expression by CD4⁺ T cells in response to S. aureus. Mononuclear cells from mammary gland lymph nodes of control mice (left panels) and immunized mice (right panels) were cultured in the presence of medium alone or heat-killed S. aureus for 72 h, and double-color flow cytometry for CD4 and IFN-γ was performed. Lymphoblasts were gated based on their forward-scatter-side-scatter profile and gated further based on CD4 expression. (A) Horizontal short lines depict the median values obtained for each group. P values were determined by using the Wilcoxon Signed Rank test for paired samples to compare the percentage of CD4⁺ T cells producing IFN-γ after stimulation with S. aureus versus medium alone (✽, P < 0.01). One representative experiment of three is shown. (B) Histograms show the intracellular IFN-γ production by CD4⁺ T cells from one representative control mouse (left) and one immunized mouse (right). Histogram curves: thick lines, cells stimulated with S. aureus; thin lines, cells cultured in medium; dotted lines, cells stained with irrelevant, isotype-matched antibodies.

FIG. 4.

IL-2 receptor (CD25) expression in T (A) and B (B) lymphocytes from regional mammary gland lymph nodes after Ima immunization with la-S. aureus. Open circles represent cells from control mice, and solid circles represent cells from immunized mice. Horizontal short lines indicate the median percentage of positive-stained cells for each group. Significance: ✽, P < 0.05; ✽✽, P < 0.01 (Mann-Whitney test).

FIG. 5.

Intracellular IFN-γ expression by CD4⁺ and CD8⁺ T cells during in vivo infection with S. aureus. Mononuclear cells from mammary gland lymph nodes of S. aureus-challenged control (○) and immunized (•) mice were obtained and triple-color flow cytometry for CD4, CD8, and IFN-γ was performed. Lymphoblasts were gated based on their forward-scatter-side-scatter profile and gated further based on CD4 (A) or CD8 (B) expression. Horizontal short lines show the median values obtained for each group. P values were determined by using the Mann-Whitney test to compare the percentage of CD4⁺ or CD8⁺ T cells producing IFN-γ in each group (✽, P < 0.05; ✽✽, P < 0.01). One representative experiment of three is shown.