Differences in innate immune responses correlate with differences in murine susceptibility to Chlamydia muridarum pulmonary infection

Abstract

We investigated the phenotypic basis for genetically determined differences in susceptibility and resistance to Chlamydia muridarum pulmonary infection using BALB/c and C57BL/6 mice. Following C. muridarum intranasal inoculation, the intensity of infection was very different between BALB/c and C57BL/6 beginning as early as 3 days post-infection. Intrapulmonary cytokine patterns also differed at early time-points (days 2 and 4) between these two strains of mice. The early recruitment of neutrophils to lung tissue was greater in BALB/c than in C57BL/6 mice and correlated with a higher number of inclusion forming units (IFU) of C. muridarum. At day 12 post-infection, BALB/c mice continued to demonstrate a greater burden of infection, significantly higher lung cytokine levels for tumour necrosis factor-alpha and interleukin-17 (IL-17) and a significantly lower level for interferon-gamma than did C57BL/6 mice. In vitro, bone-marrow-derived dendritic cells (BMDCs) from BALB/c mice underwent less functional maturation in response to C. muridarum infection than did BMDCs from C57BL/6 mice. The BMDCs of BALB/c mice expressed lower levels of activation markers (CD80, CD86, CD40 and major histocompatibility complex class II) and secreted less IL-12 and more IL-23 than BMDCs from C57BL/6 mice. Overall, the data demonstrate that the differences exhibited by BALB/c and C57BL/6 mice following C. muridarum pulmonary infection are associated with differences in early innate cytokine and cellular responses that are correlated with late differences in T helper type 17 versus type 1 adaptive immune responses.

Figure 1

Susceptibility of BALB/c mice and C57BL/6 mice following Chlamydia muridarum infection. Groups of eight mice were intranasally inoculated with 2000 inclusion-forming units (IFU) of C. muridarum and body weight changes were monitored every 2 days (a). Mice were killed at day 12 post-infection and IFU in lung tissue were titrated as described in the Materials and methods (b). The data represent the mean ± SD from eight individual mice. One of three independent trials with similar results is shown. *P < 0·05.

Figure 2

Early stage susceptibility of BALB/c mice and C57BL/6 mice following Chlamydia muridarum infection. Groups of eight mice were intranasally inoculated with 8000 inclusion-forming units (IFU) of C. muridarum and body weight changes were monitored daily up to 4 days (a). Mice were killed at days 2 or 4 post-infection and the lungs were weighed (b). Infectivity was assessed by lung IFU titration at days 2 and 4 (c). The data represent the mean ± SD from eight individual mice. One of three independent trials with similar results is shown. *P < 0·05.

Figure 3

Cytokine secretions in lung tissue of BALB/c mice versus C57BL/6 mice after infection with Chlamydia muridarum. Groups of eight mice for each strain were inoculated intranasally with 8000 inclusion-forming units (IFU; for days 2 and 4 infection) or 2000 IFU (for day 12 infection) of C. muridarum. At days 2, 4 and 12 post-infection, lung homogenates were prepared for lung IFU titrations and for assays of cytokine levels by enzyme-linked immunosorbent assay. The data represent the mean ± SD from eight individual mice. One of three independent trials with similar results is shown. *P < 0·05.

Figure 4

Cytokine production by splenocytes in response to Chlamydia muridarum stimulation. Groups of nine mice were inoculated intranasally with 2000 inclusion-forming units (IFU) of C. muridarum and splenocytes were prepared at day 12 followed by stimulation with or without heat-killed C. muridarum elementary bodies (EBs) for 72 hr. The level of C. muridarum responsive interferon-γ (IFN-γ) and interleukin-17 (IL-17) in splenocyte culture supernatants was analysed by enzyme-linked immunosorbent assay. The data represent the mean ± SE from nine individual mice. *P < 0·05.

Figure 5

Cell recruitment and neutrophil chemokine production in lung tissue of BALB/c and C57BL/6 mice after infection with Chlamydia muridarum. Groups of three mice were inoculated intranasally with 8000 inclusion-forming units (IFU) of C. muridarum and day 2 post-infection lungs were harvested, single cell suspensions were created and cell types were enumerated by fluorescence-activated cell sorter analysis. Two independent experiments were carried out. Dot-plot from one representative mouse shows double staining of surface markers for lung inflammatory cells (a). Chemokine secretion in lung tissue of BALB/c mice and C57BL/6 mice at day 2 after infection with C. muridarum. Lung homogenates were the same samples prepared for Fig. 3. The chemokines in lung homogenates were analysed by enzyme-linked immunosorbent assay. The data represent the mean ± SE from eight individual mice. One of three independent trials with similar results is shown. *P < 0·05 (b).

Figure 6

Surface phenotype and cytokine secretion of cultured dendritic cells (DCs) derived from BALB/c mice and C57BL/6 mice. Bone-marrow-derived dendritic cells (BMDCs) were generated and purified from C57BL/6 and BALB/c mice as described in the Materials and methods. Cells were infected with live Chlamydia muridarum elementary bodies at a multiplicity of infection of 3 for 24 hr and collected for staining of costimulatory molecule expression [CD80, CD86, CD40, major histocompatibility complex class II (MHC II) and Toll-like receptor 2 (TLR2)] by fluorescence-activated cell sorting (FACS). The thin-line histograms represent unstained cells as controls and bold-line histograms represent stained cells. Numerical values depict the percentage of positive cells and fluorescence intensity of relevant surface molecules. Data are representative of three independent experiments with similar results (a). Fold increase of surface molecular expression on BMDCs after C. muridarum stimulation represent mean ± SE (n = 9) of three separate experiments (b). After 48 hr incubation with or without C. muridarum, culture supernatants were collected for enzyme-linked immunosorbent assay cytokine test. The data represent the mean ± SD of three separated experiments. *P < 0·05 (c).