Age alterations in extent and severity of experimental intranasal infection with Chlamydophila pneumoniae in BALB/c mice

Abstract

The intracellular bacterium Chlamydophila ("Chlamydia") pneumoniae is a pathogen for several respiratory diseases and may be a factor in the pathogenesis of chronic diseases of aging including atherosclerosis and Alzheimer's disease. We assessed whether aging is coupled with increased burden of infection in BALB/c mice after intranasal infection by C. pneumoniae. Six- and twenty-month-old BALB/c mice were infected intranasally with 5 x 10(4) inclusion forming units (IFU) or 5 x 10(5) IFU of C. pneumoniae. Lung, brain, and heart tissue were analyzed for infectious C. pneumoniae and for Chlamydophila antigen by immunohistochemistry. At both doses, aging was associated with a decreased proportion of animals that cleared infection from the lung and greater burden of infectious organism within the lung. We observed dose-dependent spread to the heart/ascending aorta in animals infected with C. pneumoniae. In mice given 5 x 10(4) IFU, spread to the heart by day 14 was only observed in old mice. By day 28, all animals inoculated with 5 x 10(4) IFU showed evidence of spread to the heart, although higher C. pneumoniae titers were observed in the hearts from old mice. In mice inoculated with 5 x 10(5) IFU, spread of C. pneumoniae to the heart was evident by day 14, with no discernible age effect. C. pneumoniae was also recovered from the central nervous system (brain and olfactory bulb) of all mice by day 28 postinfection, with higher C. pneumoniae titers in old animals than in young animals. Our results suggest that infection with C. pneumoniae may be more severe in old animals.

FIG. 1.

Recovery of C. pneumoniae from the lung at days 14 and 28. The number of IFU of C. pneumoniae/milliliter, on a log scale, recovered from lung tissue homogenate of young and aged mice is shown on the y axis. The x axis displays the age of the animal at inoculation, the infectious dose, and the time of sacrifice. Each dot represents the results from a single animal. The bars show the arithmetic mean of all animals in each group (log₁₀). (A) Recovery of infectious C. pneumoniae at days 14 and 28 after intranasal inoculation of 5 × 10⁴ IFU. The “+” symbol indicates a statistically significant difference (P = 0.026) at day 28 p.i. between the arithmetic means of the groups of young and old mice inoculated with 5 × 10⁵ IFU. The “++” symbols indicate a statistically significant difference (P = 0.04) at day 28 p.i. between the arithmetic means of the groups of young and old mice inoculated with 5 × 10⁴ IFU. (B) Recovery of infectious C. pneumoniae at days 14 and 28 after intranasal inoculation of 5 × 10⁵ IFU.

FIG. 2.

Aged mice display a greater degree of inflammatory infiltration after infection with Chlamydophila pneumoniae. After infection with C. pneumoniae, pulmonary inflammatory infiltrates were observed in all mice. The loss of typical lung architecture, including consolidation of alveoli and inflammatory infiltrates, was observed in all infected mice, but was more prominent in aged mice. A typical lung architecture was observed in all uninfected mice at 6 (A) and 20 (B) months. Inflammatory infiltrates in young mice at 6 months (5 × 10⁴ IFU [C] and 5 × 10⁵ IFU [E]) were less severe than those in aged mice at 20 months (5 × 10⁴ IFU [D] and 5 × 10⁵ IFU [f]). Bar, 100 μm.

FIG. 3.

Chlamydophila antigen is present in the lungs of both young and aged mice at day 28 after infection. C. pneumoniae antigens were detected in the lungs of young and aged mice. Young mice display very low infectious titers at day 28 p.i., but Chlamydophila antigen was detected in the lung tissues of both young and aged mice by using C. pneumoniae-specific antibody (Dako M6600). Representative images of uninfected mice are shown in panels A (6 months) and B (20 months). Representative images of lung tissues from mice infected with C. pneumoniae, displaying immunoreactivity with C. pneumoniae-specific antibody, are shown in panels C (6 months, 5 × 10⁴ IFU), D (20 months, 5 × 10⁴ IFU), E (6 months, 5 × 10⁵ IFU), and F (20 months, 5 × 10⁵ IFU). Areas displaying C. pneumoniae immunoreactivity from panels E and F, respectively, are displayed in panels G (6 months, 5 × 10⁵ IFU) and H (20 months, 5 × 10⁵ IFU). Bar, 10 μm.

FIG. 4.

Recovery of C. pneumoniae from the heart and/or ascending aorta at days 14 and 28. The numbers of IFU of C. pneumoniae per milliliter, on a log scale, recovered from heart tissue homogenate of young and aged mice are shown on the y axis. The x axis displays the age of the animal at inoculation, the infectious dose, and the time of sacrifice. Each dot represents the results from a single animal. The bars show the arithmetic mean of each group (log₁₀). (A) Recovery of infectious C. pneumoniae at days 14 and 28 after intranasal inoculation of 5 × 10⁴ IFU. The “++” symbols indicate a statistically significant difference (P = 0.0001) at day 14 p.i. between the arithmetic means of the groups of young and old mice inoculated with 5 × 10⁴ IFU. The “+” symbol indicates a statistically significant difference (P = 0.003) at day 28 p.i. between the arithmetic means of the groups of young and old mice inoculated with 5 × 10⁴ IFU. (B) Recovery of infectious C. pneumoniae at days 14 and 28 after intranasal inoculation of 5 × 10⁵ IFU.

FIG. 5.

Chlamydophila antigens are present in heart tissue at day 28 after infection. Chlamydophila lipopolysaccharide antigens are present in heart tissue at day 28 postinfection. A majority of the immunoreactivity was localized to the endothelial cell layer. Images of heart tissue from young (6 months) mice are seen in panels A (uninfected), C (5 × 10⁴ IFU), and E (5 × 10⁵ IFU). Panels B (uninfected), D (5 × 10⁴ IFU), and F (5 × 10⁵ IFU) are representative images of aged (20 months) mice. Arrows indicate immunoreactivity for C. pneumoniae lipopolysaccharide antigens. Bar, 10 μm.

FIG. 6.

Recovery of C. pneumoniae from the brain and olfactory bulb at days 14 and 28. The numbers of IFU of C. pneumoniae per milliliter, on a log scale, recovered from brain tissue homogenate of young and aged mice are shown on the y axis. The x axis displays the age of the animal at inoculation, the infectious dose, and the time of sacrifice. Each dot represents a single animal. The bars show the arithmetic mean of all animals in each group (log₁₀). (A) Recovery of infectious C. pneumoniae at days 14 and 28 after intranasal inoculation of 5 × 10⁴ IFU. The “++” symbols indicate a statistically significant difference (P = 0.008) at day 28 p.i. between the arithmetic means of the groups of young and old mice inoculated with 5 × 10⁴ IFU. (B) Recovery of infectious C. pneumoniae at days 14 and 28 after intranasal inoculation of 5 × 10⁵ IFU. The “+” symbol indicates a statistically significant difference (P = 0.004) at day 28 p.i. between the arithmetic means of the groups of young and old mice inoculated with 5 × 10⁵ IFU.

FIG. 7.

Chlamydophila antigens are present within endothelial cells of blood vessels in the brain at day 28 after infection. Immunoreactivity for Chlamydophila antigens (Dako M6600) observed in brain tissue of C. pneumoniae-infected and mock-infected BALB/c mice at day 28 postinfection. Similar to the heart, the immunoreactivity was localized to the endothelial cells and the perivascular region. Representative images of brain tissue from young (6 months) mice are seen in panels A (uninfected), C (5 × 10⁴ IFU), and E (5 × 10⁵ IFU). Panels B (uninfected), D (5 × 10⁴ IFU), and F (5 × 10⁵ IFU) show representative images of aged (20 months) mice. Arrows indicate immunoreactivity for Chlamydophila antigens. Bar, 10 μm.