Severe Acinetobacter baumannii sepsis is associated with elevation of pentraxin 3

Abstract

Multidrug-resistant Acinetobacter baumannii is among the most prevalent bacterial pathogens associated with trauma-related wound and bloodstream infections. Although septic shock and disseminated intravascular coagulation have been reported following fulminant A. baumannii sepsis, little is known about the protective host immune response to this pathogen. In this study, we examined the role of PTX3, a soluble pattern recognition receptor with reported antimicrobial properties and stored within neutrophil granules. PTX3 production by murine J774a.1 macrophages was assessed following challenge with A. baumannii strains ATCC 19606 and clinical isolates (CI) 77, 78, 79, 80, and 86. Interestingly, only CI strains 79, 80, and 86 induced PTX3 synthesis in murine J774a.1 macrophages, with greatest production observed following CI 79 and 86 challenge. Subsequently, C57BL/6 mice were challenged intraperitoneally with CI 77 and 79 to assess the role of PTX3 in vivo. A. baumannii strain CI 79 exhibited significantly (P < 0.0005) increased mortality, with an approximate 50% lethal dose (LD50) of 10(5) CFU, while an equivalent dose of CI 77 exhibited no mortality. Plasma leukocyte chemokines (KC, MCP-1, and RANTES) and myeloperoxidase activity were also significantly elevated following challenge with CI 79, indicating neutrophil recruitment/activation associated with significant elevation in serum PTX3 levels. Furthermore, 10-fold-greater PTX3 levels were observed in mouse serum 12 h postchallenge, comparing CI 79 to CI 77 (1,561 ng/ml versus 145 ng/ml), with concomitant severe pathology (liver and spleen) and coagulopathy. Together, these results suggest that elevation of PTX3 is associated with fulminant disease during A. baumannii sepsis.

FIG 1

A. baumannii induction of PTX3 production in monocytic cells. Levels of PTX3 in J774a.1 macrophage cell supernatants were assessed, as previously described in Materials and Methods, 24 h post-challenge with several A. baumannii strains. Error bars represent ±SD for triplicate wells in all graphs. Statistical differences were determined using one-way ANOVA with Holm-Sidak correction; *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

FIG 2

Comparison of survival following challenge with A. baumannii CI 77 and 79 challenge. Mice were assessed for survival following challenge with either the PTX3-noninducing strain CI 77 or the PTX3-inducing strain CI 79, using an in vivo mouse intraperitoneal sepsis model as previously described in Materials and Methods. Mice were challenged with a high dose (designated CI 77/79 High; 10⁷ CFU; n = 8), a 100-fold-lower dose (designated CI 77/79 Low; 10⁵ CFU; n = 8), UV-inactivated bacteria (designated CI 77/79 UV; equivalent to 10⁷ CFU; n = 8) or heat-killed (HK) bacteria (designated CI 77/79 HK; equivalent to 10⁷ CFU; n = 4). Statistical differences were determined using the Mantel-Cox log rank test; ***, P < 0.0005.

FIG 3

Chemokine and PTX3 levels in A. baumannii-challenged mice. In vivo production of PTX3 and chemokines was determined as described in Materials and Methods. (A) Serum PTX3 levels were monitored over a 24-h period (n = 6) in mice challenged with A. baumannii strain CI 77 or 79. The gray bar indicates basal PTX3 production observed in the mock control group over 24 h (mean ± 1 SD). (B) Chemokines KC, MCP-1, and RANTES (mock, n = 22; CI 77, n = 24; CI 79, n = 12) were assessed 24 h postchallenge, as were serum MPO levels (n = 6). Error bars represent ±SD in all graphs; statistical differences were determined by the Welch t test; *, P < 0.05; **, P < 0.005; ***, P < 0.0005.

FIG 4

Pathology associated with A. baumannii challenge. Tissues were collected 24 h after A. baumannii low-dose (10⁵) challenge and assessed and scored for pathology by H&E staining as previously described in Materials and Methods. (A and B) 4× (A) or 10× (B) objective magnification of representative liver tissue pathology from mock-challenged (left), CI 77-challenged (center), and CI 79-challenged (right) mice. Polymorphonuclear cell infiltration and apoptotic nuclei are designated by black and white arrows, respectively. (C) Respective hepatic veins and microvessels from mock-challenged (left), CI 77-challenged (center), and CI 79-challenged (right) mice. (D) Representative spleen tissue pathology from mock-challenged (left), CI 77-challenged (center), and CI 79-challenged (right) mice. Images are representative of 3 biological samples per treatment. Abbreviations: WP, splenic white pulp; em, emboli; mv, hepatic microvessel; V, hepatic vein. Scale bars: 4×, 1 mm; 10×, 400 μm; 20×, 200 μm; 40×, 100 μm.

FIG 5

Assessing effects of A. baumannii infection on coagulation. Coagulation parameters ROTEM (EXTEM and FIBTEM), prothrombin time (PT), and ELISA for plasma D-dimer and fibrinogen determinations were performed as previously described in Materials and Methods. (A) EXTEM tracings (top) and corresponding α-angle, CF, CFT, and MCF parameters (bottom) assessing hemostatic potential of blood activated by tissue factor and phospholipid obtained from mock-challenged (n = 5), CI 77-challenged (n = 6), and CI 79-challenged (n = 4) mice. (B) FIBTEM tracings (top) and corresponding α-angle, CF, CFT, and MCF parameters (bottom) assessing the contribution of fibrin polymerization to clot formation with platelet function inhibited in mock-challenged (n = 5), CI 77-challenged (n = 6), and CI 79-challenged (n = 5) mice. (C) Clinical coagulation profiles, including PT, D-dimer, and fibrinogen assays from mock-challenged (n = 8), CI 77-challenged (n = 9), and CI 79-challenged (n = 4) mice represented in box whisker plots. Error bars represent ±SD in panels A and B. Statistical differences were determined using either the Welch t test (A and B) or the Kruskal-Wallis test with Dunn correction (C); *, P < 0.05; **, P < 0.005.