T-Cell-independent humoral immunity is sufficient for protection against fatal intracellular ehrlichia infection

Abstract

Although humoral immunity has been shown to contribute to host defense during intracellular bacterial infections, its role has generally been ancillary. Instead, CD4 T cells are often considered to play the dominant role in protective immunity via their production of type I cytokines. Our studies of highly pathogenic Ehrlichia bacteria isolated from Ixodes ovatus (IOE) reveal, however, that this paradigm is not always correct. Immunity to IOE infection can be induced by infection with a closely related weakly pathogenic ehrlichia, Ehrlichia muris. Type I cytokines (i.e., gamma interferon, tumor necrosis factor alpha, and interleukin-12) were not necessary for E. muris-induced immunity. In contrast, humoral immunity was essential, as shown by the fact that E. muris-infected B-cell-deficient mice were not protected from IOE challenge and because E. muris immunization was effective in CD4-, CD8-, and major histocompatibility complex (MHC) class II-deficient mice. Immunity was unlikely due to nonspecific inflammation, as prior infection with Listeria monocytogenes did not induce immunity to IOE. Antisera from both wild-type and MHC-II-deficient mice provided at least partial resistance to challenge infection, and protection could also be achieved following transfer of total, but not B-cell-depleted, splenocytes obtained from E. muris-immunized mice. The titers of class-switched antibodies in immunized CD4 T-cell- and MHC class II-deficient mice, although lower than those observed in immunized wild-type mice, were significant, indicating that E. muris can induce class switch recombination in the absence of classical T-cell-mediated help. These studies highlight a major protective role for classical T-cell-independent humoral immunity during an intracellular bacterial infection.

FIG. 1.

E. muris infection generates dose-dependent immunity to IOE challenge infection. a. C57BL/6 mice were infected with 4 × 10⁴ E. muris, and 30 days later infected and uninfected control mice were challenged with high-dose IOE (2× LD₅₀). Morbidity was monitored using the criteria described in Materials and Methods. Four mice were used per group. b. Bacterial infection in the spleens of immunized and control mice was determined on the indicated days postinfection. Standard deviations of the means are indicated. c. Mice were immunized with the indicated doses of E. muris and challenged with high-dose IOE. d. IOE challenge infections of immunized mice were performed using from 1 to 8 times the LD₅₀, as indicated. In all experiments at least four mice were used in each group. e. Mice were infected via the peritoneum with L. monocytogenes (Lm; 7.2 × 10⁴ CFU) 7 days prior to challenge with IOE. Control mice received only Lm or IOE, or were immunized prior to IOE challenge with E. muris (Em-IOE). f. Mice were immunized via the peritoneum with E. muris and challenged with IOE via the peritoneum (i.p.) or intravenously (i.v.). The protection observed following either route of injection was statistically significant (P < 0.04).

FIG. 2.

T cells are not essential for E. muris-induced immunity following IOE challenge. a. CD4-deficient mice were immunized with E. muris and then challenged with IOE (2× LD₅₀) approximately 30 days later. Similar studies were performed with CD8-deficient (b) and class II MHC-deficient (c) mice. Unimmunized (unimm) and immunized (imm) mice were used as controls. Data in panel b were statistically significant (P = 0.043). d. Wild-type and CD4-deficient mice were immunized with E. muris and then challenged with IOE. One group of wild-type mice was administered anti-CD4 1 day prior to IOE challenge (GK1.5; 200 μg/injection). e. Naive mice were administered 2 × 10⁶ splenocytes (imm. SPCs) or flow cytometry-purified CD4 T cells (imm. CD4⁺; 99.2% purity) that had been obtained from E. muris-immunized mice at least 30 days after immunization. All experiments utilized three to five mice per group.

FIG. 3.

B cells are essential for immunity. a. B-cell-deficient (μMT) mice were immunized with E. muris and challenged with IOE (2× LD₅₀), and morbidity was monitored. The data were statistically significant (P = 0.043; analysis of the immunized groups). b. Bacterial infection was quantitated in spleens of wild-type, B-cell-deficient (B cell KO), and CD4-deficient (CD4 KO) mice. Each data point represents a single mouse. Brackets indicate data used for statistical analysis. *, P = 0.05. n.s., not significant. c. One day prior to IOE challenge, naive mice were administered 1 × 10⁶ total (imm. SPCs) or B-cell-depleted splenocytes obtained from E. muris-immunized donor mice. Each group contained four mice. The data were statistically significant (P = 0.017).

FIG. 4.

Type I cytokines are not required for protective immunity. a. Wild-type and IFN-γ-, TNF-α-, and IL-12 p40-deficient mice were immunized with E. muris and later challenged with 2× LD₅₀ of IOE. In each case the groups of gene-targeted mice were statistically different from the wild-type control mice (P = 0.043). b. E. muris-immunized mice were treated with two doses of anti-IFN-γ neutralizing antibody (clone XMG1.2; 200 μg/dose) 1 and 4 days after IOE challenge. Naïve and immunized mice were used as controls. c. Serum concentrations of IFN-γ were measured on day 16 post-IOE challenge in the antibody-treated and nontreated immunized mice. Standard deviations are indicated. *, P < 0.05.

FIG. 5.

Immune serum can transfer protection against IOE challenge. a. Serum was pooled from immunized mice at least 30 days after infection and was administered to naïve mice 1 day prior and 3 and 7 days post-IOE challenge (100 μl/dose). Normal serum was administered as a control. Data combined from four experiments are shown (P = 0.0001; n = 17). The Ig titer of the immune sera was greater than 1:1,200. b. The monoclonal antibody Ec56.5, or an isotype control, was administered to naïve mice 1 day prior and 3 and 7 days post-IOE infection (200 μg/dose). Each group contained four mice. The data shown are representative of two experiments. c. Serum was pooled from E. muris-immunized MHC-II-deficient mice on day 30 postinfection and was administered to naive mice, as described for panel a. The differences were statistically significant (P < 0.0082; n = 5). The Ig titer of the immune serum was greater than 1:200.

FIG. 6.

T-cell-independent antibody responses in E. muris-immunized mice. OMP-19 antibody titers were measured in sera of E. muris-immunized wild-type, MHC class II-, and CD4-deficient mice. Differences in titers between wild-type and gene-targeted strains were statistically significant, except where indicated (n.s.) (Mann-Whitney test with 99% confidence interval; P < 0.05).