A new role of the complement system: C3 provides protection in a mouse model of lung infection with intracellular Chlamydia psittaci.

The complement system modulates the intensity of innate and specific immunity. While it protects against infections by extracellular bacteria its role in infection with obligate intracellular bacteria, such as the avian and human pathogen Chlamydia (C.) psittaci, is still unknown. In the present study, knockout mice lacking C3 and thus all main complement effector functions were intranasally infected with C. psittaci strain DC15. Clinical parameters, lung histology, and cytokine levels were determined. A subset of infections was additionally performed with mice lacking C5 or C5a receptors. Complement activation occurred before symptoms of pneumonia appeared. Mice lacking C3 were ∼100 times more susceptible to the intracellular bacteria compared to wild-type mice, with all C3(-/-) mice succumbing to infection after day 9. At a low infective dose, C3(-/-) mice became severely ill after an even longer delay, the kinetics suggesting a so far unknown link of complement to the adaptive, protective immune response against chlamydiae. The lethal phenotype of C3(-/-) mice is not based on differences in the anti-chlamydial IgG response (which is slightly delayed) as demonstrated by serum transfer experiments. In addition, during the first week of infection, the absence of C3 was associated with partial protection characterized by reduced weight loss, better clinical score and lower bacterial burden, which might be explained by a different mechanism. Lack of complement functions downstream of C5 had little effect. This study demonstrates for the first time a strong and complex influence of complement effector functions, downstream of C3 and upstream of C5, on the outcome of an infection with intracellular bacteria, such as C. psittaci

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<p><b>10. Anti-</b><b><i>C. psittaci</i></b><b> serum protects C3^−/− mice efficiently when transfer is performed within 1 day p.i.</b> To raise a high-titer immune serum, WT mice were infected with 4×10⁴ IFU <i>C. psittaci</i> and re-infected after 28 days. After 56 days, serum of the clinically recovered mice was collected and heat inactivated to eliminate functional donor C3. C3^−/− mice were infected with 4,000 IFU of <i>C. psittaci</i> and received anti-<i>C. psittaci</i> serum either 1 day (dashed line) or 7 days (dotted line) afterwards. Alternatively, heat-inactivated control serum from non-infected mice was transferred to the knockout mice on day 1 p.i. (solid line). Survival rates were determined for up to 42 days post-infection. * indicates significant differences (p<0.05) between mice which received the anti-<i>C. psittaci</i> serum as compared to the mice which received control serum, and ** or *** p<0.01 and p<0.001, respectively.</p

Figure

<p><b>6. Bacterial load, histological score and MPO in C3^−/− and WT mice in low-dose </b><b><i>C. psittaci</i></b><b> infection.</b> Mice were infected with 200 IFU of <i>C. psittaci</i> DC15 or mock-infected and sacrificed on day 21 p.i. Bacterial load (panel a), histological score (panel b) and MPO (panel c) in C3^−/− and WT mice in low-dose <i>C. psittaci</i> infection were determined. The figure shows data obtained from <i>C. psittaci</i>-infected mice (cps, gray bars), mock-infected mice (white bars), WT mice (open bars) and C3^−/− (horizontally hatched bars). Means ± SEM are depicted. * indicates significant (p<0.05) differences between infected knockout as compared to the corresponding infected BL/6J WT mice.</p

Figure

<p><b>10. Anti-</b><b><i>C. psittaci</i></b><b> serum protects C3^−/− mice efficiently when transfer is performed within 1 day p.i.</b> To raise a high-titer immune serum, WT mice were infected with 4×10⁴ IFU <i>C. psittaci</i> and re-infected after 28 days. After 56 days, serum of the clinically recovered mice was collected and heat inactivated to eliminate functional donor C3. C3^−/− mice were infected with 4,000 IFU of <i>C. psittaci</i> and received anti-<i>C. psittaci</i> serum either 1 day (dashed line) or 7 days (dotted line) afterwards. Alternatively, heat-inactivated control serum from non-infected mice was transferred to the knockout mice on day 1 p.i. (solid line). Survival rates were determined for up to 42 days post-infection. * indicates significant differences (p<0.05) between mice which received the anti-<i>C. psittaci</i> serum as compared to the mice which received control serum, and ** or *** p<0.01 and p<0.001, respectively.</p

Figure

<p><b>4. No indication of lack of cytokine response in the absence of a functional complement system.</b> Mice were infected with 10⁴ IFU of <i>C. psittaci</i> DC15 or mock-infected. The cytokines IFN-γ, TNF-α, IL-6 and IL-10 (panel a-d) were determined in the lung homogenate of the animals sacrificed on day 4 or 9. The figure shows data obtained from <i>C. psittaci</i>-infected mice (cps, gray bars), mock-infected mice (white bars), WT mice (open bars) and C3^−/− (horizontally hatched bars). * indicates significant (p<0.05) differences between infected knockout as compared to the corresponding infected BL/6J WT mice. Means ± SEM are depicted. * indicates significant (p<0.05) differences between infected knockout mice as compared to the corresponding infected BL/6J WT mice.</p

Figure

<p><b>5. Illness with delayed onset in C3^−/− mice in low-dose </b><b><i>C. psittaci</i></b><b> lung infection.</b> C3^−/− and the corresponding BL/6J WT mice were either infected with 200 IFU of <i>C. psittaci</i> DC15 (<i>cps</i>; filled symbols) or mock-infected (<i>mock</i>; open symbols). Body weight (a) and clinical score (b) were determined daily for up to 28 days. Seventy % of the C3^−/− mice survived low-dose infection, as did all WT mice (c). The means ± SEM are depicted. * indicates significant differences (p<0.05) between infected knockout mice as compared to the corresponding infected BL/6J WT mice.</p

2way ANOVA for effect of <i>C. psittaci</i> DC15 infection: p<0.001.

<p>2way ANOVA for effect of <i>C. psittaci</i> DC15 infection: p<0.001.</p

Figure

<p><b>2. Bacterial load, histological score and MPO of C3^−/− and WT mice in high-dose infection.</b> Mice were infected with 10⁴ IFU of <i>C. psittaci</i> DC15 or mock-infected and sacrificed on days 4 or 9 p.i. Bacterial load (panel a), histological score (panel b) and granulocyte marker enzyme MPO (panel c) in lungs of C3^−/− and WT mice were determined. The figure shows data obtained from <i>C. psittaci</i>-infected mice (cps, gray bars), mock-infected mice (white bars), WT mice (open bars) and C3^−/− (horizontally hatched bars). Means ± SEM are depicted. * indicates significant (p<0.05) differences between infected knockout as compared to the corresponding infected BL/6J WT mice.</p

Figure

<p><b>8. Bacterial load in spleen of infected C3^−/− and WT mice.</b> Mice were infected with 10⁴ IFU of <i>C. psittaci</i> DC15 and sacrificed on days 4 or 9 p.i. Bacterial load in spleen of C3^−/− (hatched columns) and WT mice (open columns) were determined. A similar pattern as compared to lung homogenate became apparent. However, the differences in bacterial load between infected C3^−/− and WT mice were not significant. In spleen of mock-infected mice, no chlamydiae were detected. Means ± SEM are depicted.</p

Figure

<p><b>1. C3 is deleterious during early </b><b><i>C. psittaci</i></b><b> lung infection, but protective in the late phase.</b> C3^−/− and the corresponding BL/6J wild-type (WT) mice were either intranasally infected with 10⁴ IFU of <i>C. psittaci</i> DC15 (<i>cps</i>; filled symbols) or mock-infected (<i>mock</i>; open symbols) (Fig. 1a and b). In panel c, varying amounts of <i>C. psitttaci</i> (10⁴, 2000 or 200 IFU per mouse) were applied as indicated. Body weight (a), clinical score (b) and survival rates (c) were determined for up to 21 days post-infection. Depicted are the means ± SEM. * indicates significant differences (p<0.05) between infected C3^−/− as compared to the corresponding infected BL/6J WT mice, and ** or *** p<0.01 and p<0.001, respectively.</p