Delayed clearance of Ehrlichia chaffeensis infection in CD4+ T-cell knockout mice

Abstract

Human monocytic ehrlichiosis is an emerging tick-borne disease caused by the rickettsia Ehrlichia chaffeensis. To examine the role of helper T cells in host resistance to this macrophage-tropic bacterium, we assessed E. chaffeensis infections in three mouse strains with differing functional levels of helper T cells. Wild-type, C57BL/6J mice resolved infections in approximately 2 weeks. Major histocompatibility complex class II (MHCII) knockout, B6.129-Abb(tm1) mice lacking helper T cells developed persistent infections that were not resolved even after several months. CD4+ T-cell-deficient, B6.129S6-Cd4(tm1Knw) mice cleared the infection, but the clearance took 2 weeks longer than it did for wild-type mice. C57BL/6J mice resolved infection more rapidly following a second experimental challenge, but B6.129S6-Cd4(tm1Knw) mice did not. The B6.129S6-Cd4(tm1Knw) mice also developed active E. chaffeensis-specific immunoglobulin G responses that were slightly lower in concentration and slower to develop than that observed in C57BL/6J mice. E. chaffeensis-specific cytotoxic T cells were not detected following a single bacterial challenge in any mouse strain, including wild-type C57BL/6J mice. However, the cytotoxic T-cell activity developed in all three mouse strains, including the MHCII and CD4+ T-cell knockouts, when challenged with a second E. chaffeensis infection. The data reported here suggest that the cell-mediated immunity, orchestrated by CD4+ T cells is critical for conferring rapid clearance of E. chaffeensis.

FIG. 1.

Distribution of CD4⁺ and CD8⁺ T cells in B6, CD4D, and C2D mouse thymuses and spleens. Spleen cells and thymocytes were stained with PE-Cy5-conjugated anti-CD3ɛ, PE-conjugated anti-CD8, and fluorescein isothiocyanate-conjugated anti-CD4 monoclonal antibodies. Cells were gated on CD3-positive cells, and profiles of CD4⁺ and CD8⁺ cells are shown for the following tissue samples: B6 thymus and spleen, C2D thymus and spleen, and CD4D thymus and spleen.

FIG. 2.

Comparison of clearance of E. chaffeensis by B6, CD4D, and C2D mice after single and dual E. chaffeensis challenges. The figure shows detection of E. chaffeensis in peritoneal exudate cells (A to D) or spleen (E and F) by RT-PCR (C to F) or culture techniques (A and B) after a single (A, C, and E) or secondary (B, D, and F) experimental challenge. Numbers represent percent mice positive for B6 (▵), C2D (○), or CD4D (•) mice, four to six mice per time point.

FIG. 3.

Liver lesion severity in mice infected with E. chaffeensis. Lesions were evaluated at various time intervals and assigned lesion scores (described in Materials and Methods). Data are presented as the mean scores. Error bars represent standard deviations. Analysis included four mice per each postinfection date.

FIG. 4.

Kinetics of a secondary CTL response. The figure shows the CTL response of B6 (top), CD4D (middle), and C2D (bottom) mice against uninfected (left) and E. chaffeensis-infected (right) targets at 3, 6, 8 (B6 mice only), and 13 days after a secondary experimental E. chaffeensis challenge. Numbers represent the mean CTL responses of cells from four mice per time point assayed independently, except for the 8-day B6 data, which were from two mice.

FIG. 5.

IgG subclass distribution in B6 and CD4D mice for E. chaffeensis infection. Plasma samples from all postinfection day mice were analyzed by quantitative ELISA using purified whole-cell antigen. The data are presented by day postinfection and are pooled from four mice each from all postinfection dates. Each bar represents the median IgG concentration determined from samples analyzed.

FIG. 6.

Western blot profile showing response to E. chaffeensis whole-cell antigen. Representative data for each postinfection date following a single bacterial injection are presented. Plasma samples were analyzed 7, 10, 13, 15, 17, 21, and 24 days postinfection.