The infecting dose of Chlamydia muridarum modulates the innate immune response and ascending infection

Abstract

Murine vaginal infection with the obligate intracellular bacterium Chlamydia muridarum is commonly used as a model for ascending Chlamydia infections of the human female genital tract. Gamma interferon-producing Th1 cells, in concert with other mononuclear infiltrates, primarily mediate antichlamydial immunity. However, many factors modify this response, including the bacterial load. To investigate the manner in which the inoculating dose of C. muridarum modulates a genital infection, we measured innate and adaptive cell numbers, CD4+ lymphocyte cytokine profile, chemokine expression, course of infection, and pathological sequelae in genital tracts of BALB/c mice infected with doses of C. muridarum ranging from 10(4) to 10(7) inclusion-forming units. We found that the influx of both innate and adaptive immune cells responded similarly in the lower genital tract (cervical-vaginal tissues) and upper genital tract (oviduct tissues) to increasing inoculating doses. However, cells expressing the innate markers Gr-1 and CD11c were affected to a greater degree by increasing dose than lymphocytes of the adaptive immune response (Th1, CD4+, CD8+, CD19+), resulting in a change in the balance of innate and adaptive cell numbers to favor innate cells at higher infecting doses. Surprisingly, we detected greater numbers of viable chlamydiae in the oviducts at lower inoculating doses, and the number of organisms appeared to directly correlate with hydrosalpinx formation after both primary infection and repeat infection. Taken together, these data suggest that innate immune cells contribute to control of ascending infection.

FIG. 1.

Effect of dose on the distribution of CD4⁺ and IFN-γ-producing Th1 cells within the genital tract. (A) FACS profiles of CD4⁺ cells in CV and OD tissues 7 days postinfection from one representative experiment. Percentages of CD4⁺ cells were calculated from lymphocytes gated by forward- and side-scatter analysis. (B) Number of CD4⁺ cells in CV and OD tissues per 10⁶ genital tract cells during the onset of adaptive immunity (days 7 and 14). Data are compiled from two to three independent experiments. There were statistically greater numbers of CD4⁺ cells on day 14 compared to day 7 for all doses (*, P = 0.01 by two-way ANOVA). (C) Chlamydia-responsive Th1 cells were identified by intracellular cytokines IFN-γ (+) and IL-4 (−) within gated CD4⁺ CD69⁺ lymphocytes. (D) Number of Th1 cells in CV and OD tissues per 10⁶ genital tract cells. Data are compiled from two independent experiments.

FIG. 2.

Effect of dose on innate and adaptive immunity following secondary challenge with C. muridarum. Leukocytes were isolated from pools of collagenase-digested genital tracts of 20 mice, infected with increasing doses of C. muridarum, and reinfected 50 days later with the original dose, 6 days post-secondary infection. We used immunofluorescence and flow cytometry to identify polymorphonuclear cells (Gr-1⁺), dendritic cells (CD11c⁺), monocytes/macrophages (CD14⁺), and CD4⁺, CD8⁺, and CD19⁺ cells in two separate experiments. Data are the means and standard errors of the means for the number of positive cells per 10⁶ genital tract cells. There was no statistical difference in the magnitude or distribution of any cell type in the genital tract with dose upon secondary infection.

FIG. 3.

Course of infection by vaginal swabs and tissue homogenates following genital inoculation with increasing Chlamydia dose. Vaginal swabs and tissue homogenates of cervical-vaginal regions, uterine horns, and oviducts were collected following primary and secondary inoculations of mice with increasing Chlamydia doses for recovery of viable organisms. Swab data are the means and standard errors of the means of log₁₀ IFU ml⁻¹ for n = 9 mice from two independent experiments (y axis, right); homogenate data are the means and standard errors of the means of log₁₀ IFU/mg of tissue for 5 to 10 mice from two or three independent experiments (y axis, left). Shedding of chlamydiae from genital tracts of mice infected with the highest dose (10⁷ IFU) was significantly greater during the resolution phase of primary and secondary infections than that with lower doses. +, P < 0.05 compared to results for doses of 10⁴ IFU (days 9 and 12) and 1.5 × 10⁵ IFU (day 12) by two-way repeated-measure ANOVAs; *, P < 0.001 compared to results for doses of 10⁴ and 1.5 × 10⁵ IFU (days 53 and 56) by two-way repeated-measure ANOVAs. In contrast, when chlamydiae were cultured from homogenized tissues, there was no difference in the magnitude of infection within a given region (two-way ANOVA), though there was an effect between regions. #, P < 0.05 for results with OD tissue compared to those with CV and uterine tissue by two-way ANOVA. 10⁷ IFU, days 7, 14, and 21; 1.5 × 10⁵ IFU and 10⁴ IFU, day 7.

FIG. 4.

Inducible protein expression of the CXCR3 ligand CXCL10/IP-10 in the genital tract with increasing Chlamydia dose. CXCL10/IP-10 protein was detected by ELISA in OD and CV tissue homogenates of individual mice infected with increasing Chlamydia inocula, 7 and 14 days post-primary infection and 6 days post-secondary infection. Data are expressed relative to total homogenate protein and are the means and standard errors of the means for two independent experiments with five mice per dose per experiment. There was no effect of Chlamydia dose on increased CXCL10/IP-10 in OD tissues compared to CV tissues after primary infection or on the magnitude or kinetics of CXCL10/IP-10 induction following primary and secondary infections. However, at higher doses there was a statistical increase in the distribution between OD and CV tissues 7 days after primary infection (1 × 10⁷ and 1.5 × 10⁵ IFU; *, P < 0.05 by two-way ANOVA).

FIG. 5.

Oviduct dilation in mice following infection with increasing Chlamydia dose. Oviduct diameters were measured from H&E-stained sections collected transversally at the ovary-to-oviduct transition of three to eight mice from one or two independent experiments during (A) primary infection (day 49) and (B) 6 days post-repeat infection. Data are expressed as the average number of grids (one grid equals approximately 1 μm) of two diameter measurements per oviduct section of four sections per oviduct. There was no statistical difference in oviduct diameter for increasing dose during primary or secondary infection by one-way ANOVA.