Yersinia enterocolitica promotes robust mucosal inflammatory T-cell immunity in murine neonates

Abstract

Mucosal immunity to gastrointestinal pathogens in early life has been studied only slightly. Recently, we developed an infection model in murine neonates using the gastroenteric pathogen Yersinia enterocolitica. Here, we report that oral infection of neonatal mice with low doses of virulent Y. enterocolitica leads to vigorous intestinal and systemic adaptive immunity. Y. enterocolitica infection promoted the development of anti-LcrV memory serum IgG1 and IgG2a responses of comparable affinity and magnitude to adult responses. Strikingly, neonatal mesenteric lymph node CD4(+) T cells produced Yersinia-specific gamma interferon (IFN-gamma) and interleukin-17A (IL-17A), exceeding adult levels. The robust T- and B-cell responses elicited in neonates exposed to Y. enterocolitica were associated with long-term protection against mucosal challenge with this pathogen. Using genetically deficient mice, we found that IFN-gamma and CD4(+) cells, but not B cells, are critical for protection of neonates during primary Y. enterocolitica infection. In contrast, adults infected with low bacterial doses did not require either cell population for protection. CD4-deficient neonatal mice adoptively transferred with CD4(+) cells from wild-type, IFN-gamma-deficient, or IL-17AF-deficient mice were equally protected from infection. These data demonstrate that inflammatory CD4(+) T cells are required for protection of neonatal mice and that this protection may not require CD4-derived IFN-gamma, IL-17A, or IL-17F. Overall, these studies support the idea that Y. enterocolitica promotes the development of highly inflammatory mucosal responses in neonates and that intestinal T-cell function may be a key immune component in protection from gastrointestinal pathogens in early life.

FIG. 1.

Neonatal mice show prolonged resistance to orogastric Y. enterocolitica infection and prolonged antigen persistence compared to adult mice. Seven-day-old neonatal (NEO) and adult (AD) BALB/c mice were infected orogastrically with 5 × 10⁷ CFU (A), 5 × 10⁶ CFU (B), or 2 × 10⁵ CFU (C) of Y. enterocolitica. Survival curves were generated by Kaplan-Meier Survival analysis. The survival curves depict pooled data from three experiments (10 neonates and 14 adults) (A), two experiments (9 neonates and 10 adults) (B), or 6 experiments with 78 neonates and 51 adults (C). The survival kinetics were compared using the Mantel-Haenszel log rank test. n.s., not significant; *, P ≤ 0.046. (D) Fecal pellets from individual mice infected orogastrically with 2 × 10⁵ CFU of Y. enterocolitica (Ye) were collected at 19 days p.i. and enriched for the presence of Y. enterocolitica bacteria. Each symbol represents the percentage of mice with stools positive for Y. enterocolitica from five separate experiments with 5 to 13 neonates per experiment (total of 48 mice) and 3 to 9 adults per experiment (total of 31 mice). Symbols on the dashed line are from experiments with samples with undetectable bacteria, and the line indicates the mean for the pooled data.

FIG. 2.

APC in the MLN of infected mice express activation and costimulatory markers. Neonatal and adult BALB/c mice were infected orogastrically with 5 × 10⁵ CFU of Y. enterocolitica. (A) MLN from individual control (dashed line) and infected (solid line) mice were collected at 15 days p.i., and cells stained were with anti-CD11c, anti-CD80, or anti-CD86 antibody and analyzed by flow cytometry. The expression levels of CD80 and CD86 within CD11c⁺ cells were overlaid for each age group. The range in the MFIs of CD80 or CD86 for each group (n = 3 to 6 mice per group) is shown within each graph. (B) At 21 days p.i., MLN cells from mice infected with 2 × 10⁵ CFU of Y. enterocolitica were stained with anti-IgM and anti-MHC-II antibodies. A representative dot plot profile of IgM⁺ MHC-II⁺ cells is shown and the MFI of MHC-II within IgM⁺ cells for control (□) and infected (▪) mice is shown in panel B (right). The data from two experiments were pooled for five to seven uninfected controls and six infected neonates or four infected adults. Differences were analyzed by the Mann-Whitney test. *, P = 0.017; **, P = 0.0095.

FIG. 3.

Infection of neonatal mice with Y. enterocolitica leads to development of greater primary Yersinia-specific serum antibody responses and to secondary antibody responses similar in quantity and quality to adult responses. (A to C) BALB/c neonates and adults were infected orogastrically with 2 × 10⁵ CFU of Y. enterocolitica. (A) Serum levels of Yersinia-specific IgG1 and IgG2a antibodies using purified MBP-LcrV were measured at 19 to 20 days p.i. from individual uninfected (○) and infected (•) neonates and adults using serum samples diluted 1/150. OD₄₅₀ values are shown in the graphs (n = 7 to 11 controls and 11 to 20 infected mice). In the experiments shown in panels B and C, mice initially infected as neonates (NEO) or adults (AD) were boosted orogastrically at 9 to 11 weeks p.i. with 5 × 10⁶ CFU of Y. enterocolitica, and sera were collected at 21 days postboost and serially diluted. (B) The relative levels of IgG1 and IgG2a for 9 neonates (•) and 10 adults (○) were pooled for analysis from two independent experiments. Undetectable antibody titers were plotted at the dashed line. (C) The avidity of memory IgG1 antibodies to LcrV for 7 neonates and 6 adults was tested in a serum ELISA using increasing amounts of KSCN (0.1 to 3.5 M). The amount of KSCN that reduced the OD₄₅₀ by 50% is defined as the avidity index. Differences were calculated by the Mann-Whitney test. n.s., not significant; *, P ≤ 0.03; **, P = 0.007.

FIG. 4.

Mice infected orogastrically as neonates are protected against challenge in adulthood with a high dose of Y. enterocolitica. Neonatal and adult BALB/c mice were infected orogastrically with 2 × 10⁵ CFU of Y. enterocolitica. Nine to 11 weeks p.i., mice were orogastrically infected with 2 × 10⁸ CFU of Y. enterocolitica in parallel with age-matched naïve mice. (A) Weight loss was used as a surrogate marker for mortality; mice that lost over 25% of their body weight were deemed moribund. The bar graphs show the percent survival at 7 days postchallenge of naïve age-matched and primed mice pooled from three independent experiments (17 to 21 mice per group). (B) At 7 days postchallenge, the PP from naïve and primed mice (n = 5 to 6 mice per group) were analyzed for Y. enterocolitica titers. The dotted line is at the limit of detection. Differences were analyzed by the Mann-Whitney test. n.s., not significant; *, P ≤ 0.03 between naïve and primed mice in each group.

FIG. 5.

The inflammatory cytokines IFN-γ and IL-17A are produced by MLN cells from infected neonates upon restimulation in vitro with Yersinia antigens. (A and C) Neonatal and adult BALB/c mice were infected orogastrically with 2 × 10⁵ CFU of Y. enterocolitica. (A) At 19 to 20 days p.i., individual MLN were harvested from three to four control and eight to nine infected mice, and 4 × 10⁵ MLN cells were cultured in vitro with 10 μg/well of HKY for 72 h. The amounts of IFN-γ, IL-17A, and IL-4 quantified by ELISA from three experiments are shown, with the horizontal line indicating the mean. (B) Neonatal and adult C57BL/6 mice were infected orogastrically with 1 × 10⁷ CFU of Y. enterocolitica. At 20 days p.i., MLN cells from individual mice were cultured as described in panel A. (C) At 20 to 21 days p.i., MLN CD4⁺ T cells were isolated from BALB/c mice, and 2 × 10⁵ cells were stimulated with 4 × 10⁵ splenic APC pulsed with HKY for 72 h. IFN-γ, IL-17A, and IL-4 were quantified by ELISA. The pooled data from three experiments are shown in the bar graphs. (D) The fold difference in IFN-γ and IL-17A levels between age-matched control and infected mice was calculated from each experiment in panel C and pooled for analysis (nine infected neonates and eight infected adults). Fold difference values greater than 1 are above the dotted line. In all experiments, differences between groups were analyzed by the Mann-Whitney test. n.s., not significant; *, P ≤ 0.035; **, P ≤ 0.0048; ***, P ≤ 0.002.

FIG. 6.

The levels of CD4⁺ Foxp3⁺ T cells in the MLN of infected adults are increased compared to infected neonates, and their presence affects cytokine production in vitro. (A to C) Neonatal and adult BALB/c mice were infected orogastrically with 2 × 10⁵ CFU of Y. enterocolitica. (A) MLN were harvested from control and infected mice at 21 days p.i.; total MLN or purified CD4⁺ cells were stained with anti-CD4 and anti-Foxp3 antibodies. The percentage of CD4⁺ cells expressing Foxp3 was quantified by flow cytometry. Each dot represents the percentage of CD4⁺ Foxp3⁺ cells from three to four individual mice in each group or CD4⁺ cells pooled from two to four mice. Differences were analyzed by the Mann-Whitney test. n.s., not significant; *, P = 0.016; **, P = 0.0047. (B) At 21 to 23 days p.i., CD4⁺ MLN cells were isolated, followed by depletion of CD25⁺ cells using monoclonal 7D4 antibody. CD4⁺ cells were stained with anti-CD25 and anti-Foxp3 antibodies before and after CD25 depletion. A representative flow cytometry profile from an infected adult is shown with the percentage ± SEM of CD25⁺ Foxp3⁺ cells from six samples in each age group. (C) Total CD4⁺ and CD4⁺ CD25⁻ cells were cultured in vitro with splenic APC pulsed with HKY. Supernatants were collected 72 h after culture, and the levels of IFN-γ and IL-17A were quantified by ELISA. The fold change in cytokine production between CD4⁺ CD25⁻ cells and total CD4⁺ cells for each age group is shown, and the horizontal line indicates the mean from two independent experiments (n = 6 in each group). Fold difference values greater than 1 are above the dotted line. Differences in fold change were analyzed by the Mann-Whitney test. n.s., not significant; **, P = 0.0043.

FIG. 7.

Neonatal mice deficient in CD4⁺ cells (CD4^−/−) and IFN-γ (IFN-γ^−/−) are more susceptible to orogastric infection, while the absence of B cells only modestly increases their susceptibility to Y. enterocolitica. (A and B) Wild-type C57BL/6 and μMT neonates and adults were infected orogastrically with 1 × 10⁷ CFU of Y. enterocolitica. The percent survival from three experiments for each age group is depicted with a connecting line (n = 2 to 6 neonates and n = 3 to 6 adults of each strain per experiment). Differences between groups were analyzed by an unpaired t test. n.s., not significant. (B) The data in panel A were used to generate survival curves for neonates (10 wild-type and 13 μMT mice) and adults (11 wild type and 18 μMT mice). Survival curves were analyzed by the Mantel-Haenszel log rank test. n.s., not significant. (C) Wild-type C57BL/6 and CD4^−/− neonatal and adult mice were infected orogastrically with 1 × 10⁷ CFU of Y. enterocolitica (left) or 1 × 10⁸ CFU of Y. enterocolitica (right). The percent survival from three experiments is connected with a line. Differences between groups were analyzed by an unpaired t test. *, P = 0.04. (D) The data shown in panel C were pooled for neonates (17 wild-type and 14 CD4^−/− mice) and adults (15 wild-type and 20 CD4^−/− mice) and analyzed by the Mantel-Haenszel log rank test. n.s., not significant; **, P = 0.0052. (E) Neonatal and adult C57BL/6 (wild type)and IFN-γ^−/− mice were infected orogastrically with 1.4 × 10⁷ CFU of Y. enterocolitica. Survival was recorded from two experiments with 7 wild-type and 16 IFN-γ^−/− neonates and 11 wild-type and 13 IFN-γ^−/− mice adults. (F) Adult mice were also infected with 3 × 10⁸ CFU of Y. enterocolitica, and survival curves were generated from 10 wild-type and 13 IFN-γ^−/− mice. Data were analyzed by the Mantel-Haenszel log rank test. n.s., not significant; **, P = 0.0014; ***, P = 0.0005.

FIG. 8.

Adoptively transferred wild-type, IFN-γ^−/− or IL-17AF^−/− CD4⁺ T cells improve survival of neonatal CD4^−/− mice following infection with Y. enterocolitica, and MLN cells from these mice produce inflammatory cytokines upon in vitro stimulation with Y. enterocolitica antigens. Lymph node CD4⁺ T cells (6 × 10⁶) from adult C57BL/6, IFN-γ^−/−, or IL-17AF^−/− mice were injected intravenously into 1-day-old CD4^−/− mice. (A) Seven-day-old wild-type C57BL/6, CD4^−/−, CD4^−/− mice injected with wild type CD4⁺ cells, CD4^−/− mice injected with IFN-γ^−/− CD4⁺ cells, and CD4^−/− mice injected with IL-17AF^−/− CD4⁺ cells were infected orogastrically with 1 × 10⁷ CFU of Y. enterocolitica. The percent survival values from two to three individual experiments are connected with a line. (B) The data from the experiments in panel A were pooled to generate survival curves and analyzed by the Mantel-Haenszel log rank test. n.s., not significant; *, P ≤ 0.03; **, P = 0.0025 (for CD4^−/− versus CD4^−/− mice injected with CD4⁺ cells). (C) The small intestines were collected at 25 days p.i. from infected wild-type, CD4^−/−, CD4^−/− mice injected with IFN-γ^−/− CD4⁺ cells, or CD4^−/− mice injected with IL-17AF^−/− CD4⁺ cells, and tissues analyzed for Yersinia titers. The dashed line is at the limit of detection. The data from two experiments were pooled (n = 7 to 9 mice per group) and analyzed by the Mann-Whitney test. n.s., not significant; *, P = 0.01; **, P = 0.0012. (D) MLN from mice that survived the infection at 21 to 25 days p.i. were collected, and cell suspensions were stained with anti-CD4 and anti-CD44 antibodies. Representative flow cytometry profiles show the percentages of CD4⁺ (top) and CD44⁺ cells within CD4⁺ cells (bottom), with the range of cells from three to four experiments (n = 5 to 17 mice). (E) MLN cells from individual mice described in panel D were cultured for 72 h with 10 μg/well of HKY. MLN cells from uninfected C57BL/6 (n = 8), CD4^−/− (n = 25), and CD4^−/− mice injected with wild-type CD4⁺ cells (n = 5) were stimulated along with cells from infected CD4^−/− (n = 15), CD4^−/− mice injected with wild-type CD4⁺ cells (n = 8), CD4^−/− mice injected with IFN-γ^−/− CD4⁺ cells (n = 17), and CD4^−/− mice injected with IL-17AF^−/− CD4⁺ cells (n = 9). Supernatants were tested for IFN-γ and IL-17A by ELISA. Differences were analyzed by the Mann-Whitney test. n.s., not significant; For IL-17A, significance is indicated as follows: *, P ≤ 0.013 between infected CD4^−/− plus IL-17AF^−/− CD4⁺ cells versus infected wild-type or infected CD4^−/− mice; **, P ≤ 0.0049, ***P ≤ 0.0004 (between selected groups). For IFN-γ, significance is indicated as follows: **, P ≤ 0.0025 between infected CD4 ^−/− plus IFN-γ^−/− CD4⁺ cells versus infected wild-type or infected CD4^−/− mice; ***, P ≤ 0.0004 (betwen selected groups).