Effects of repeated Chlamydia pneumoniae inoculations on aortic lipid accumulation and inflammatory response in C57BL/6J mice

Abstract

Chlamydia pneumoniae is a common respiratory tract pathogen, and persistent infections have been associated with atherosclerosis. We studied the effects of repeated chlamydial inoculations on the inflammatory response and on aortic lipid accumulation in C57BL/6J mice. Mice fed a diet supplemented with 0.2% cholesterol were infected three or six times with C. pneumoniae every fourth week. Sera and lungs were analyzed for inflammatory responses, lung tissues were tested for the presence of C. pneumoniae DNA and RNA, and intimal lipid accumulation in the aortic sinus was quantified. High levels of chlamydial heat shock protein 60 (Hsp60) immunoglobulin G2c subclass antibodies were detected in all of the infected mice, and a positive and statistically significant correlation was found between these antibodies and autoantibodies against mouse Hsp60. Both Hsp60 antibody levels correlated with the severity of lung tissue inflammation. The cholesterol supplement in the diet had no effect on serum cholesterol levels. Significantly larger intimal lipid lesions were seen in the mouse group infected six times (6,542 mum(2)) than in the control group (1,376 mum(2); P = 0.034). In conclusion, repeated inoculations increased aortic sinus lipid accumulation in normocholesterolemic mice. The correlation between the antibodies to mouse and chlamydial Hsp60 proteins and their association with lung inflammation further support the theory of the development of an autoimmune response against heat shock proteins after repeated chlamydial infections.

FIG. 1.

Total C. pneumoniae IgG antibody levels measured by the MIF and EIA methods and levels of mouse Hsp60 IgG antibodies measured by EIA. The study group, the number of inoculations given (e.g., six inoculations [6x]), and the age at which the first inoculation was given (e.g., 8 weeks) are indicated. Cpn, C. pneumoniae; chol., cholesterol; w, weeks; Geom., geometric.

FIG. 2.

Spearman's correlation between chlamydial Hsp60 IgG2c and mouse Hsp60 IgG levels in all mice (A) and in mice at 32 weeks of age (B).

FIG. 3.

(A) C. pneumoniae DNA positivity in lung tissue samples measured by nested PCR. (B) Lung inflammatory reactions measuring perivascular and peribronchial mononuclear cell infiltrates on a scale from 0 (no inflammation) to 4 (severe infiltration with occasional alveolar macrophages). Group I, six inoculations, starting at the age of 8 weeks; group II, three inoculations, starting at the age of 8 weeks; group III, three inoculations, starting at the age of 20 weeks; group IV, three inoculations, starting at the age of 20 weeks, and cholesterol feeding started at 18 weeks; group V, uninfected control. w, weeks.

FIG. 4.

Lipid lesion areas in the aortic sinuses of the mice (and geometric means for the groups) inoculated with C. pneumoniae. Samples were taken at the age of 32 weeks. Group I, six inoculations, starting at the age of 8 weeks; group II, three inoculations, starting at the age of 8 weeks; group III, three inoculations, starting at the age of 20 weeks; group IV, three inoculations, starting at the age of 20 weeks, and cholesterol feeding started at 18 weeks; group V, uninfected control. One asterisk and two asterisks indicate significant differences compared with group I (P = 0.034 and P = 0.045, respectively). geom., geometric.