Innate immune responses to systemic Acinetobacter baumannii infection in mice: neutrophils, but not interleukin-17, mediate host resistance

Abstract

Acinetobacter baumannii is a nosocomial pathogen with a high prevalence of multiple-drug-resistant strains, causing pneumonia and sepsis. The current studies further develop a systemic mouse model of this infection and characterize selected innate immune responses to the organism. Five clinical isolates, with various degrees of antibiotic resistance, were assessed for virulence in two mouse strains, and between male and female mice, using intraperitoneal infection. A nearly 1,000-fold difference in virulence was found between bacterial strains, but no significant differences between sexes or mouse strains were observed. It was found that microbes disseminated rapidly from the peritoneal cavity to the lung and spleen, where they replicated. A persistent septic state was observed. The infection progressed rapidly, with mortality between 36 and 48 h. Depletion of neutrophils with antibody to Ly-6G decreased mean time to death and increased mortality. Interleukin-17 (IL-17) promotes the response of neutrophils by inducing production of the chemokine keratinocyte-derived chemoattractant (KC/CXCL1), the mouse homolog of human IL-8. Acinetobacter infection resulted in biphasic increases in both IL-17 and KC/CXCL1. Depletion of neither IL-17 nor KC/CXCL1, using specific antibodies, resulted in a difference in bacterial burdens in organs of infected mice at 10 h postinfection. Comparison of bacterial burdens between IL-17a(-/-) and wild-type mice confirmed that the absence of this cytokine did not sensitize mice to Acinetobacter infection. These studies definitely demonstrate the importance of neutrophils in resistance to systemic Acinetobacter infection. However, neither IL-17 nor KC/CXCL1 alone is required for effective host defense to systemic infection with this organism.

Fig. 1.

Bacterial kinetics in organs of mice infected i.p. with A. baumannii strain 4502. (A) C57BL/6 mice challenged with a 6-LD₅₀ dose; (B) C3HeB/FeJ mice challenged with a 0.5-LD₅₀ dose. Inoculating doses are indicated by arrows on the y axis. Animals were euthanized at 4, 8, 12, 16, and 24 h postinfection, and livers, lungs, spleens, and blood samples were harvested and plated to determine the number of CFU per gram of tissue or per 0.1 ml of blood. Points represent individual animals. Lines represent median values for bacterial burdens.

Fig. 2.

Bacterial kinetics in organs of mice infected i.p. with A. baumannii strain 5798. (A) C57BL/6 mice challenged with a 1-LD₅₀ dose; (B) C3HeB/FeJ mice challenged with a 0.2-LD₅₀ dose. Inoculating doses are indicated by arrows on the y axis. Animals were euthanized at 4, 8, 12, and 16 h postinfection, and livers, lungs, spleens, and blood samples were harvested and plated to determine the number of CFU per gram of tissue or per 0.1 ml of blood. Points represent individual animals. Lines represent median values for bacterial burdens. #, counts > 10⁴ CFU/ml.

Fig. 3.

Neutrophils are important for host innate immune response to systemic A. baumannii infection. C3HeB/FeJ mice were injected i.p. with 25 μg of either anti-Ly6G (neutrophil-specific) or control IgG2b antibody 24 h prior to, at the time of, and 24 h after i.p. injection with a given dose of A. baumannii strain 4502. Survival was monitored for 7 days. Numbers in parentheses represent number dead/total. *, P < 0.0001 for survival; **, P < 0.05 for survival distribution based on the log rank test.

Fig. 4.

In vitro induction of IL-17 from peritoneal exudate cells by recombinant IL-23 and LPS. PECs were collected from naive C3HeB/FeJ mice or from mice infected with a 1.2-LD₅₀ dose of A. baumannii strain 4502. Cells plated at the given concentrations were treated with recombinant IL-23 (A) or LPS (B). Culture supernatants were harvested 24 h or 48 h after stimulation and analyzed for IL-17 production by ELISA.

Fig. 5.

Kinetics of IL-17, KC/CXCL1, and MCP-1 induction following Acinetobacter infection. C3HeB/FeJ mice were infected i.p. with A. baumannii strain 4502 in 4 experiments, with doses ranging between 1.1 and 2.3 LD₅₀s, or injected i.p. with saline. Peritoneal exudate fluid was harvested at the indicated time points and analyzed for IL-17 (pg/ml) (A) or KC/CXCL1 (ng/ml) (B) by ELISA. Points represent duplicate samples, 5 to 15 samples per time point. *, P < 0.05 for infected versus saline; **, P < 0.0001 for infected versus saline.

Fig. 6.

Depletion of IL-17 or KC/CXCL1 by antibody neutralization does not sensitize C3HeB/FeJ mice to Acinetobacter infection. C3HeB/FeJ mice were injected i.p. with either anti-IL-17 MAb (A), anti-KC/CXCL1 MAb (B), or control Ig2a MAb 1 h before i.p. infection with a 1-LD₅₀ dose (A) or a 2-LD₅₀ dose (B) of A. baumannii 4502. Inoculating doses are indicated by arrows on the y axis. Mice were euthanized for necropsy at 10 h postinfection. Points represent individual animals. Lines represent median values for bacterial burdens. Levels of organisms in mice treated with anti-17 or anti-KC/CXCL1 are not statistically significantly different from levels in mice given control antibody in any organ at either challenge dose.

Fig. 7.

IL-17A knockout mice are not sensitized to A. baumannii. IL-17a^−/− and wild-type C57BL/6 mice were infected i.p. with a 17-LD₅₀ dose (A) or a 0.6-LD₅₀ dose of A. baumannii strain 4502. Inoculating doses are indicated by arrows on the y axis. Mice were euthanized for necropsy at 8 h (A) or 10 h (B) postinfection. Points represent individual animals. In panel B, filled symbols represent male mice, and open symbols represent female mice. Lines represent median values for bacterial burdens. Levels of organisms in any organ or blood sample of IL-17a^−/− mice are not statistically significantly different from the level for WT mice.