Abstract
Rationale: α2-Antiplasmin (A2AP) is a major inhibitor of fibrinolysis by virtue of its capacity to inhibit plasmin. Although the fibrinolytic system is strongly affected by infection, the functional role of A2AP in the host response to sepsis is unknown.
Objectives: To study the role of A2AP in melioidosis, a common form of community-acquired sepsis in Southeast Asia and Northern Australia caused by the gram-negative bacterium Burkholderia pseudomallei.
Methods: In a single-center observational study A2AP was measured in patients with culture-proven septic melioidosis. Wild-type and A2AP-deficient (A2AP−/−) mice were intranasally infected with B. pseudomallei to induce severe pneumosepsis (melioidosis). Parameters of inflammation and coagulation were measured, and survival studies were performed.
Measurements and Main Results: Patients with melioidosis showed elevated A2AP plasma levels. Likewise, A2AP levels in plasma and lung homogenates were elevated in mice infected with B. pseudomallei. A2AP-deficient (A2AP−/−) mice had a strongly disturbed host response during experimental melioidosis as reflected by enhanced bacterial growth at the primary site of infection accompanied by increased dissemination to distant organs. In addition, A2AP−/− mice showed more severe lung pathology and injury together with an increased accumulation of neutrophils and higher cytokine levels in lung tissue. A2AP deficiency further was associated with exaggerated systemic inflammation and coagulation, increased distant organ injury, and enhanced lethality.
Conclusions: This study is the first to identify A2AP as a protective mediator during gram-negative (pneumo)sepsis by limiting bacterial growth, inflammation, tissue injury, and coagulation.
α2-Antiplasmin (A2AP) is a major inhibitor of fibrinolysis; however, the functional role of A2AP in the host response to sepsis is unknown.
The present study is the first to describe the role of A2AP during severe gram-negative sepsis (melioidosis). Patients with severe gram-negative sepsis (melioidosis) show elevated A2AP plasma levels. During experimental melioidosis, A2AP functions as a protective mediator by limiting inflammation, tissue injury, coagulation, and bacterial growth.
During severe infection and sepsis, a range of host defense mechanisms becomes activated, resulting in a strong proinflammatory response together with hemostatic alterations (1–3). These changes, characterized by activation of coagulation and inhibition of fibrinolysis, can be regarded as host protective in containing the causative microorganisms and the associated inflammation to the site of the infection, rendering this relationship physiologically effective (1, 3). Too much procoagulant activity, however, can also be disadvantageous due to excess fibrin formation, which may block capillary flow and consequently induce organ failure, and by influencing inflammatory activity, leading to disproportionate inflammation in, for example, the alveolar compartment during pneumonia (1). Increased inflammation and the accompanying disseminated intravascular coagulation are important causes of multiorgan failure and mortality during sepsis (1, 2, 4).
The serpin α2-antiplasmin (A2AP) is considered one of the main inhibitors of fibrinolysis (5, 6). The regulatory role of A2AP in fibrinolysis consists of formation of irreversible complexes with plasmin (PAP complexes [PAPc]), formation of reversible complexes with plasminogen that inhibit adsorption of plasminogen to fibrin, and making fibrin more resistant to local plasmin activity through cross-linking via factor XIIIa (FXIIIa) (7). Besides its role in fibrinolysis, no other clear functions for A2AP are known. Plasma A2AP levels were found elevated in patients with acute stroke, myocardial infarction, unstable angina, and atrial fibrillation, suggesting a self-defense system against complications of ischemic events (8). Patients with severe sepsis showed unaltered, elevated, or decreased plasma A2AP concentrations when compared with healthy control subjects (9–12). Several studies have consistently documented elevated circulating PAPc levels in patients with sepsis, suggestive for a functional role of A2AP in inhibiting fibrinolysis during severe infection (12–14). Notably, the plasma levels of plasminogen activator inhibitor type I (PAI-1), the other main inhibitor of fibrinolysis, were elevated in patients with severe pneumonia and sepsis (15–19), which in experimental pneumosepsis models in mice contributed to protective immunity (20–22) by mechanisms that at least in part were unrelated to its inhibitory effects on fibrinolysis (20–23). In contrast, to the best of our knowledge, the functional role of A2AP in sepsis has not been studied previously.
In the present study we sought to identify the role of A2AP during melioidosis, a severe septic disease caused by the gram-negative bacillus Burkholderia pseudomallei. Melioidosis is characterized by pneumonia and rapid bacterial dissemination to distant body sites, with many possible disease manifestations, septic shock being the most severe (24–26). Melioidosis is an important cause of community-acquired sepsis in Southeast Asia and Northern Australia, with mortalities up to 40% despite appropriate antibiotic therapy (24–26). Additionally, B. pseudomallei is recently classified as a Tier 1 disease agent considered to be an exceptional threat to security (27). Recently, we revealed that during melioidosis fibrinolysis might hamper the antibacterial host response: tissue-type plasminogen activator (tPA)-deficiency was associated with host protective effects (28), whereas PAI-1 deficiency led to a detrimental phenotype (22). We here identify A2AP as a possible protective mediator during gram-negative (pneumo)sepsis caused by B. pseudomallei.
Thirty-four patients (mean age, 52 yr; range, 18–86 yr) with sepsis caused by B. pseudomallei and 32 healthy control subjects (mean age, 41 yr; range, 21–59 yr) from the same area were studied. All subjects were recruited prospectively at Sapprasithiprasong Hospital, Ubon Ratchathani, Thailand. Sepsis due to melioidosis was defined as culture positivity for B. pseudomallei from any clinical sample plus a systemic inflammatory response syndrome (2, 29). Study design and subjects have been described in detail (29). The study was approved by the Ministry of Public Health, Royal Government of Thailand and the Oxford Tropical Research Ethics Committee, University of Oxford, Oxford, UK. Written informed consent was obtained from all subjects before the study.
Pathogen-free 10-week-old male wild-type (WT) C57BL/6 mice were purchased from Charles River (Maastricht, The Netherlands) and maintained at the animal care facility of the Academic Medical Center (University of Amsterdam), according to national guidelines, with free access to food and water. A2AP−/− mice (30) were a kind gift from Dr. H. R. Lijnen (Center for Molecular and Vascular Biology, KU Leuven, Belgium) and were backcrossed for at least eight times on a C57BL/6 genetic background. All experiments were approved by the Animal Care and Use Committee of the Academic Medical Center, Amsterdam, The Netherlands.
Experimental melioidosis was induced by intranasal inoculation with B. pseudomallei strain 1026b (300 cfu/50 μl 0.9% NaCl) as previously described (22, 28, 29). For survival experiments, mice were checked every 6 hours until death occurred. Sample harvesting, determination of bacterial growth, and assays, including Western blot, are described in the online supplement.
Human data are expressed as scatter dot plots with medians. Data of mice experiments are expressed as box-and-whisker plots showing the smallest observation, lower quartile, median, upper quartile, and largest observation or as medians with interquartile ranges. Comparisons between groups were tested using the Mann-Whitney U test. For survival studies, Kaplan-Meier analyses followed by log-rank tests were performed. All analyses were done using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA). P values less than 0.05 were considered statistically significant.
To obtain insight into A2AP expression during melioidosis, we measured A2AP protein levels in plasma from 34 patients with sepsis with culture-proven B. pseudomallei infection and 32 local healthy control subjects. Fourteen (41%) patients with melioidosis died in the hospital. A2AP was elevated in patients with melioidosis with median plasma concentrations approximately 1.4-fold higher than in healthy subjects (Figure 1A; median 81% in control subjects vs. 113% in patients, P < 0.0001). No differences in A2AP were found between survivors and nonsurvivors (data not shown).
Figure 1. α2-Antiplasmin (A2AP) concentrations during clinical and experimental melioidosis. Plasma A2AP levels in patients with culture-proven melioidosis (A). Increased levels of plasma A2AP in patients with sepsis (n = 34, open squares) compared with healthy control subjects (n = 32, black squares). Data are expressed as scatter dot plots showing all observations and the median. ***P < 0.0001 for patients versus controls (Mann-Whitney U test). Endogenous A2AP levels during experimental melioidosis. Wild-type (WT) mice were killed before (uninfected), 24, 48, and 72 hours after intranasal inoculation with Burkholderia pseudomallei. A2AP protein concentrations were measured in plasma (B) and lung homogenates (C), n = 8 mice per group at each time point. Data are expressed as box-and-whisker plots showing the smallest observation, lower quartile, median, upper quartile, and largest observation. *P < 0.05, **P < 0.01, and ***P < 0.001 versus uninfected (Mann-Whitney U test).
[More] [Minimize]To study the role of A2AP during melioidosis at the tissue level, mice were intranasally inoculated with live B. pseudomallei to induce pneumonia-derived melioidosis (22, 28, 29) and killed after 24, 48, and 72 hours. Infection with B. pseudomallei was associated with a significant increase in plasma A2AP levels at 24, 48, and 72 hours after inoculation (P < 0.05 vs. baseline 24 and 48 h after inoculation, P < 0.01 72 h after inoculation; Figure 1B) and a significant increase in lung A2AP levels 24 and 48 hours after inoculation (P < 0.05 at 24 h and P < 0.001 at 48 h after inoculation; Figure 1C).
To investigate whether A2AP deficiency impacts pulmonary bacterial growth, we determined bacterial loads in lung homogenates. Although during the early stage of infection pulmonary bacterial counts were similar in A2AP−/− and WT mice, 48 and 72 hours after inoculation A2AP−/− mice demonstrated markedly elevated bacterial loads in their lungs (P < 0.01 after 48 h, P < 0.001 after 72 h; Figure 2A). To determine whether A2AP deficiency impacts bacterial dissemination, we measured bacterial loads in liver and spleen homogenates and blood in A2AP−/− mice and compared them to WT mice (Figures 2B–2D). Although after 24 hours bacterial loads were similar at distant body sites of A2AP−/− and WT mice, after 48 and 72 hours an increased number of A2AP−/− mice had become bacteremic when compared with WT mice: after 48 hours, 100% of A2AP−/− mice versus 50% of WT mice had positive blood cultures, whereas after 72 hours 88% of A2AP−/− mice versus 50% of WT mice had positive blood cultures. When compared with WT mice, bacterial loads in blood were higher as well in the A2AP−/− mice (P < 0.05 and P < 0.01 after 48 and 72 h, respectively; Figure 2B). Bacterial loads in liver and spleen homogenates were reflective of those in blood: A2AP−/− mice displayed an increased bacterial dissemination to liver and spleen when compared with WT mice (P < 0.05 and P < 0.001 for bacterial loads in both liver and spleen homogenates after 48 and 72 h, respectively; Figures 2C–2D). Thus, A2AP deficiency facilitates growth (at the primary site of infection) and dissemination of B. pseudomallei, in particular at later time points.
Figure 2. α2-Antiplasmin (A2AP) deficiency is associated with increased pulmonary bacterial growth and dissemination. Mice were inoculated with 300 cfu of Burkholderia pseudomallei and killed after 24, 48, or 72 hours. cfus were determined in lung homogenates (A), blood (B), liver (C), and spleen (D) homogenates. Data are expressed as box-and-whisker plots showing the smallest observation, lower quartile, median, upper quartile, and largest observation. Gray boxes represent wild-type (WT) mice, and white boxes represent A2AP−/− mice (n = 8 mice per group for each time point). *P < 0.05, **P < 0.01, and ***P < 0.001 for the difference between WT and A2AP−/− mice (Mann-Whitney U test). BC+ = number of positive blood cultures.
[More] [Minimize]Both WT and A2AP−/− mice infected with B. pseudomallei showed inflammatory infiltrates in the lungs characterized by interstitial inflammation together with necrosis, endothelialitis, bronchitis, edema, thrombi, and pleuritis (Figures 3A–3C). At all time points, the extent of lung inflammation was significantly greater in A2AP−/− mice when compared with control mice (P < 0.01 for 24 and 72 h after infection, P < 0.05 48 h after infection; Figure 3A). Additionally, 24 and 48 hours after infection, A2AP−/− mice displayed significantly higher granulocyte counts in their lungs when compared with WT mice (P < 0.05 at 24 h postinfection; P < 0.001 at 48 h postinfection; Figures 3D–3F). Enhanced neutrophil influx into lung tissue of A2AP−/− mice was confirmed by elevated lung myeloperoxidase levels in A2AP−/− mice (Figure 3G). In accordance with enhanced pulmonary inflammation, A2AP−/− mice displayed strongly elevated concentrations of cytokines (tumor necrosis factor [TNF]-α, IL-6, IL-10, IFN-γ) and the CXC chemokine KC in whole lung homogenates (Table 1), especially at later time points after infection. As in vitro studies have shown that A2AP induces transforming growth factor (TGF)-β1 (31), we wondered how A2AP deficiency would impact active TGF (aTGF)-β1 levels during infection with B. pseudomallei. Therefore, we determined aTGF-β1 levels in lung homogenates 24, 48, and 72 hours after infection. Clearly, after 24 and 72 hours of infection, significantly lower aTGF-β1 levels were measured in A2AP−/− mice compared with WT (P < 0.05 and < 0.01, respectively; Table 1). Because the most prominent differences between mouse strains were seen after 72 hours of infection, we performed a bronchoalveolar lavage (BAL) at this time point in an independent experiment to determine whether the proinflammatory and lung injurious effects could also be detected in the alveolar compartment. Indeed, our data showed a similar increase in bacterial loads in BAL fluid (BALF) of A2AP−/− mice when compared with WT mice (P < 0.001; Figure 4A), which was accompanied by elevated protein levels, reflecting protein leakage across the bronchoalveolar barrier (P < 0.001; Figure 4B) and higher levels of lactate dehydrogenase (LDH), a general parameter for cell damage (P < 0.001; Figure 4C). In accordance, BALF cytokine and KC levels were higher in A2AP−/− mice compared with WT mice (see Table E1 in the online supplement).
Figure 3. α2-Antiplasmin (A2AP) deficiency exaggerates lung pathology and neutrophil accumulation after infection with Burkholderia pseudomallei. A2AP−/− mice infected with B. pseudomallei showed increased histopathology scores at 24, 48, and 72 hours, characterized by interstitial inflammation together with necrosis, endothelialitis, bronchitis, edema, thrombi, and pleuritis (A). Representative photographs of lungs at 72 hours postinoculation from wild-type (WT) (B) and A2AP−/− mice (C) (hematoxylin and eosin staining ×100). Increased granulocyte influx, as reflected by the intensity of Ly-6G immunostaining of histopathological slides, after 24 and 48 hours in A2AP−/− mice infected with B. pseudomallei (D). The intensity of the Ly-6G staining is expressed as the percentage of the total lung surface area. Representative photographs of Ly-6G immunostaining (original magnification ×100) for granulocytes of WT (E) and A2AP−/− (F) mice showing significantly increased neutrophil influx 48 hours postinoculation. Increased levels of pulmonary myeloperoxidase (MPO) (G), reflecting the total pulmonary neutrophil content, were seen 48 and 72 hours after infection in the lungs of A2AP−/− mice compared with WT mice. Data are expressed as box-and-whisker plots showing the smallest observation, lower quartile, median, upper quartile, and largest observation. Gray boxes represent WT mice, and white boxes represent A2AP−/− mice (n = 8 mice per group for each time point). *P < 0.05, **P < 0.01, and ***P < 0.001 for WT versus A2AP−/− mice (Mann-Whitney U test).
[More] [Minimize]| Lung Homogenates | Plasma | |||
|---|---|---|---|---|
| WT | A2AP−/− | WT | A2AP−/− | |
| 24 h | ||||
| TNF-α | 1,193 (980–1,563) | 1,663 (1,182–2,174) | 3.4 (2.2–3.9) | 2.8 (2.0–4.2) |
| IFN-γ | 3.6 (1.8–6.6) | 9.2 (6.9–14)* | 5.2 (2.5–12) | 20 (14–25)† |
| IL-6 | 1,227 (783–2,728) | 3,761 (1,727–5,372)* | 134 (52–2,860) | 154 (95–186) |
| IL-10 | 2.8 (1.8–3.4) | 2.8 (2.0–3.7) | < | < |
| IL-12p70 | 3.1 (2.1–6.60) | 7.6 (5.9–11.4) | 3.9 (3.0–5.3) | 5.9 (4.1–6.5) |
| KC, ng/ml | 18 (12–23) | 18 (11–22) | ND | ND |
| aTGF-β1, ng/ml | 2.5 (1.7–3.0) | 1.6 (0.5–2.0)* | ND | ND |
| 48 h | ||||
| TNF-α | 3,484 (2,900–4,104) | 6,881 (5,159–9,424)† | 5.2 (4.3–10) | 12 (13–17)† |
| IFN-γ | 24 (12–33) | 22 (14–45) | 69 (40–169) | 427 (260–675)† |
| IL-6 | 629 (274–826) | 1,582 (829–2,639)† | 54 (26–1,030) | 195 (140–316)† |
| IL-10 | 2.2 (1.7–3.4) | 8.7 (6.7–12.2)* | < | < |
| IL-12p70 | 36 (17–39) | 27 (16–50) | 3.8 (3.4–4.7) | 10 (8–14)† |
| KC, ng/ml | 5.8 (3.6–7.0) | 11 (7.8–11)* | ND | ND |
| aTGF-β1, ng/ml | 1.4 (1.4–3.5) | 1.6 (1.2–1.9) | ND | ND |
| 72 h | ||||
| TNF-α | 2,338 (1,379–4,040) | 9,271 (8,880–12,909)‡ | 7.8 (4.8–12) | 79 (65–184)‡ |
| IFN-γ | 7.3 (5.9–11) | 21 (14–23)† | 28 (19–119) | 250 (157–519)† |
| IL-6 | 175 (93–551) | 5,859 (3,356–6,467)‡ | 54 (26–114) | 2,024 (1,345–4,918)† |
| IL-10 | 11 (3–21) | 225 (109–281)‡ | 1.9 (90–2.7) | 18 (13–37)† |
| IL-12p70 | 8 (7–12) | 11 (9–15) | 4.2 (3.6–6.1) | 6.7 (4.1–11) |
| KC, ng/ml | 5.5 (3.4–9.7) | 27 (22–48)‡ | ND | ND |
| aTGF-β1, ng/ml | 2.1 (1.9–2.3) | 0.6 (0.5–1.3)† | ND | ND |
Figure 4. α2-Antiplasmin (A2AP) deficiency induces increased bacterial growth in the intraalveolar compartment accompanied by lung injury. Bacterial loads in bronchoalveolar lavage fluid (BALF) (A) and levels of total protein (B), lactate dehydrogenase (LDH) (C), and keratinocyte-derived chemokine (KC) (D) were increased in BALF 72 hours after intranasal inoculation with 300 cfu of Burkholderia pseudomallei in A2AP−/− mice compared with wild-type (WT) mice. Data are expressed as box-and-whisker plots showing the smallest observation, lower quartile, median, upper quartile, and largest observation. Gray boxes represent WT mice, and white boxes represent A2AP−/− mice (n = 8 mice per group for each time point). ***P < 0.001 for WT versus A2AP−/− mice (Mann-Whitney U test).
[More] [Minimize]Despite their robust inflammatory response (Table 1 and Figures 3 and 4), A2AP−/− mice exhibited increased bacteremia. Therefore, we performed a killing assay to find out whether defects in bacterial killing by infected macrophages would be responsible for these observations. Our results show no differences in bacterial killing by A2AP−/− and WT macrophages (Figure 5A). In accordance, recombinant A2AP did not have a direct growth inhibitory effect on B. pseudomallei in vitro (data not shown). Because cytokine data suggest that deficiency or the presence of A2AP might have an effect on inflammatory mediator generation by lung immune cells, we stimulated A2AP−/− and WT lung and bone marrow–derived macrophages ex vivo with heat-killed B. pseudomallei and in addition determined the impact of recombinant A2AP (Figures 5B and 5C). Our data show that neither deficiency nor addition of A2AP influenced TNF-α release by macrophages.
Figure 5. α2-Antiplasmin (A2AP) deficiency does not influence bacterial killing or cytokine release from macrophages. Bacterial killing by bone marrow–derived macrophages (BMDM). No differences between wild-type (WT) (gray dots; n = 4) and A2AP−/− mice (white dots; n = 4) in bacterial killing 1 and 6 hours after infection (A). Data are expressed as medians with interquartile ranges. Tumor necrosis factor (TNF)-α release from alveolar macrophages (AM) (B) and BMDM (C) after stimulation with heat-killed Burkholderia pseudomallei (1 × 107/ml) while exogenous A2AP (70 μg/ml) or RPMI1640 was added. After 20 hours of stimulation, no differences in TNF-α release were observed between macrophages of WT mice (gray boxes), WT mice to which exogenous A2AP was added (gray striped boxes), and A2AP−/− mice (white boxes). Data are expressed as box-and-whisker plots with medians, n = 8 mice per strain.
[More] [Minimize]As A2AP−/− mice showed increased bacterial dissemination to blood, liver, and spleen, we wondered whether this would result in increased systemic inflammation and distant organ injury. Indeed, plasma levels of cytokines were much higher in A2AP−/− than in WT mice (Table 1). In addition, the plasma levels of LDH, a marker for general cellular injury, were elevated in A2AP−/− mice relative to WT mice at 72 hours after infection with B. pseudomallei (P < 0.001; Figure 6A). At this stage of severe sepsis, A2AP−/− mice also showed increased plasma levels of aspartate transaminase and alanine aminotransferase, markers of hepatocellular injury (P < 0.001 for aspartate transaminase, P < 0.01 for alanine aminotransferase; Figures 6C and 6D, respectively). No differences in creatinine (Figure 6B) or urea (data not shown), markers for renal failure, were seen between A2AP−/− and WT mice at any of the time points. Taken together, A2AP seems to play a protective role in the development of distant organ injury during experimental melioidosis. Finally, as A2AP−/− mice showed markedly increased pulmonary and distant bacterial growth and tissue damage, we investigated whether this would influence mortality during murine melioidosis. Clearly, A2AP−/− mice demonstrated an accelerated and enhanced mortality when compared with WT mice (P < 0.01), indicating that endogenous A2AP is protective during experimental melioidosis (Figure 6E).
Figure 6. α2-Antiplasmin (A2AP) deficiency is associated with increased systemic inflammation, (hepato)cellular injury, and lethality during melioidosis. Mice were intranasally inoculated with 300 cfu of Burkholderia pseudomallei and killed after 24, 48, or 72 hours. When compared with wild-type (WT) mice, A2AP−/− mice showed enhanced distant organ injury as reflected by elevated plasma lactate dehydrogenase (LDH) levels (A) and increased hepatocellular damage as reflected by increased levels of aspartate transaminase (C), and alanine aminotransferase (D). No significant differences in plasma creatinine, a parameter for renal insufficiency, were seen in A2AP−/− mice when compared with WT mice (B). Data are expressed as box-and-whisker plots showing the smallest observation, lower quartile, median, upper quartile, and largest observation. Gray boxes represent WT mice, and white boxes represent A2AP−/− mice (n = 8 mice per group for each time point). **P < 0.01 and ***P < 0.001 for the difference between WT and A2AP−/− mice (Mann-Whitney U test). A2AP−/− mice showed an accelerated and increased mortality during experimental melioidosis (E). Mortality was assessed every 6 hours; n = 15–16 mice per group. Comparison between groups was done by Kaplan-Meier analysis followed by log-rank tests.
[More] [Minimize]TATc, a parameter of coagulation induction, was measured in plasma, lung homogenates, and BALF of WT and A2AP−/− mice 24, 48, and 72 hours after inoculation of B. pseudomallei. At the later time points, A2AP−/− mice demonstrated increased plasma TATc when compared with WT mice (P < 0.01 for both 48 and 72 h after inoculation; Figure 7A). No differences were seen in TATc concentrations in lung homogenates (Figure 7B); however, in BALF 72 hours after infection, TATc was markedly increased (P < 0.001; Figure 7C). To obtain insight into activation of fibrin degradation during infection with B. pseudomallei, fibrin degradation products (FDP), including d-dimer and fragment X levels, were measured by Western blotting. After 24 hours, no differences in FDP were seen between WT and A2AP−/− mice (Figure 7D). However, after 48 and 72 hours, FDP were markedly increased in A2AP−/− mice both in lung homogenates and in BALF (Figures 7E–7G). At these time points, d-dimer levels in lung homogenates of A2AP−/− mice demonstrated a 2.6- and 2.7-fold increase, respectively, when compared with WT mice (Figure 7H; both P < 0.01), whereas fragment X levels demonstrated a 2.8- and 3.8-fold increase, respectively (Figure 7I; both P < 0.01). In BALF, 72 hours after infection, d-dimer and fragment X levels showed a 7.5- and 7-fold increase in A2AP−/− mice, respectively (Figures 7H and 7I; both P < 0.01).
Figure 7. α2-Antiplasmin (A2AP) deficiency results in enhanced coagulation and fibrinolysis during melioidosis. Mice were infected intranasally with 300 cfu Burkholderia pseudomallei to induce melioidosis. After 48 and 72 hours, coagulation activation as reflected by levels of TATc were significantly increased in A2AP−/− mice compared with wild-type (WT) mice in plasma (A) and bronchoalveolar lavage fluid (BALF) (C), but not in lung homogenates (B). Western blotting on fibrin(ogen) degradation products including fragment X (240–260 kD) and d-dimer (200 kD) in lung homogenates and BALF 24, 48, and 72 hours after infection (D–G). When compared with WT mice, increased fibrin(ogen) degradation products were seen in A2AP−/− mice in lung homogenates 48 (E) and 72 hours (F) after infection and in BALF 72 hours after infection (G). Semiquantitative analysis of the blots showed that after 48 and 72 hours, d-dimer levels were 2.7- and 2.8-fold increased, respectively, in lung homogenates and 7.5-fold increased in BALF (H). Levels of fragment X in lung homogenates showed 2.8- and 3.8-fold increases after 48 and 72 hours, respectively, and a 7-fold increase in BALF after 72 hours (I). (C) Positive control sample (fully clotted mouse plasma, uninfected, dilution 1:50). Data are expressed as box-and-whisker plots (A–C) showing the smallest observation, lower quartile, median, upper quartile, and largest observation and as bars (H–I), showing medians with interquartile ranges. Gray boxes and gray bars represent WT mice; white boxes and white bars represent A2AP−/− mice (n = 8–9 mice per group for each time point). In panels D–G every lane represents one mouse sample (n = 6–7 mice per group for each time point). **P < 0.01 and ***P < 0.001 for the difference between WT and A2AP−/− mice (Mann-Whitney U test).
[More] [Minimize]Although several studies have described altered circulating A2AP levels in patients with severe infection (9–12), the functional role of this major fibrinolysis inhibitor in the host response to sepsis has not been investigated before. Our data clearly show that endogenous A2AP is protective during experimental gram-negative (pneumo)sepsis caused by B. pseudomallei.
A2AP is considered the principal inhibitor of plasmin and thereby one of the major inhibitors of fibrinolysis (5–7). Patients with severe pneumonia, sepsis, or ARDS were reported to have increased plasma concentrations of A2AP (9, 15), which is in line with our data. Notably, however, some investigations failed to show higher A2AP levels in patients with sepsis (10–12), in spite of the fact that severe infection, including melioidosis, invariably is associated with elevated plasma levels of PAPc, in which A2AP is irreversibly bound to plasmin (11, 12, 32, 33). Infection of WT mice with B. pseudomallei resulted in an increase in both plasma and lung A2AP levels, comparable to the human disease. Although these observational data on plasma A2AP concentrations in patients and mice with gram-negative sepsis provide a further rationale to study the involvement of A2AP in severe infection, they do not offer insight into a possible functional role, either protective or detrimental, in the host response. For this we used A2AP−/− mice. These animals have a normal phenotype, with no effect on fertility, growth, development, or post-traumatic bleeding; as expected, they do exhibit enhanced endogenous fibrinolytic activity (30).
Ample evidence has demonstrated an altered hemostatic balance during severe (pneumo)sepsis both in the lungs and the circulation, with increased procoagulant activity and inhibition of fibrinolysis (1, 34). It is considered that a disturbed fibrin turnover may promote extravascular alveolar fibrin deposition in the lung and contribute to disseminated intravascular coagulation and subsequently to respiratory distress and multiorgan failure. Also in patients with melioidosis, concurrent activation and inhibition of fibrinolysis have been described, indicated by elevated plasma levels of tPA and PAI-1, respectively, with a net state of decreased fibrinolysis, as reflected by an increased ratio between coagulation and fibrinolysis (TATc/PAPc ratio) (33). In these patients, increased TATc/PAPc ratios were also associated with a higher mortality (33), suggesting that fibrinolysis plays a role in the host defense against B. pseudomallei. In addition to these clinical data, the protective effect of fibrin during melioidosis was supported by recently published data from our laboratory using the experimental model of infection with B. pseudomallei in mice. PAI-1−/− mice, which are expected to have decreased fibrin depositions, showed a profibrinolytic state, with increased bacterial growth and inflammation, and an increased lethality (22), whereas tPA−/− mice, which are expected to have increased fibrin depositions, demonstrated lower FDP levels together with reduced bacterial growth and dissemination, less inflammation, and a lower mortality (28). Our present data are fully in line with these observations. We here demonstrate that during experimental melioidosis, A2AP deficiency is associated with a profibrinolytic state, as reflected by increased levels of FDP, including d-dimer and fragment X. This profibrinolytic state, which was most prominent after 48 and 72 hours of infection, was associated with increased release of proinflammatory cytokines, increased amounts of neutrophils in the lungs, and increased organ failure. Clearly, all these results point to a protective role for fibrin in the host response against B. pseudomallei. Fibrin may serve to “wall off” the primary infection and to prevent bacterial dissemination, which in turn may slow down progression of inflammation. In the absence of inhibitors of fibrinolysis, such as in A2AP−/− or PAI-1−/− mice, bacteria are less limited in multiplication and dissemination, which consequently may aggravate the course of infection in terms of inflammation and injury. Interestingly, A2AP deficiency also resulted in enhanced activation of coagulation, as reflected by increased levels of plasma and lung TATc. Taking into account that A2AP does not influence thrombin generation, these observations are most likely explained by the strongly increased bacterial loads in A2AP−/− mice, resulting in a more potent stimulus for the coagulation system. At later stages of infection, progressive clotting might have further contributed to multiorgan failure and increased mortality, as has been described in patients with sepsis (1, 3).
Previous experiments with A2AP−/− mice focused on the role of A2AP in production of TGF-β and fibrosis and systemic sclerosis (31, 35). TGF-β, a pleiotropic cytokine, is regarded as a key regulator of the immune response (36) but also as the major inducer of fibrosis (37). In a model of bleomycin-induced dermal fibrosis in mice, A2AP deficiency attenuated TGF-β1 production, hampered the induction of myofibroblast differentiation, and diminished fibrosis (31, 35). Moreover, it was found that A2AP specifically induced the production of TGF-β1 in fibroblasts (31, 35). Our data showing decreased levels of TGF-β1 in lung homogenates of A2AP−/− mice are in line with these observations. Interestingly, a recent study from our laboratory showed that mice infected with B. pseudomallei and treated with anti–TGF-β antibodies displayed a subtle but protective phenotype, with diminished bacterial loads in lungs and spleen and less distant organ failure; anti–TGF-β treatment did not impact mortality (38). Thus, the effect of A2AP on TGF-β levels is unlikely to contribute to its protective effect in experimental melioidosis.
Once the immune system is activated during melioidosis, neutrophils are recruited to the site of infection. Neutrophils play an important role in the host defense against B. pseudomallei, as they are involved in early bacterial containment (26, 39). On the other hand, exaggerated neutrophil recruitment and subsequent release of proinflammatory mediators may be detrimental, illustrating the “double-edged sword” character of innate immunity. Our data show that the early influx of neutrophils into lungs of A2AP−/− mice was similar to that in WT mice, arguing against a direct involvement of A2AP herein. At later time points, A2AP−/− mice displayed higher neutrophil numbers, most likely as a consequence of the higher bacterial loads. In support of this notion, A2AP−/− macrophages showed unaltered TNF-α release on exposure to Burkholderia in vitro, and recombinant A2AP did not influence macrophage responsiveness, indicating that A2AP does not directly impact inflammatory responses elicited by B. pseudomallei. This aggravated neutrophil influx, together with the release of neutrophil-derived proteases and proinflammatory cytokines, probably at least in part was responsible for the enhanced lung injury in A2AP−/− mice, as reflected by lung histopathology scores and elevated protein and LDH concentrations in BALF. A2AP−/− mice in addition showed increased distant organ damage, including hepatocellular injury, reflected by elevated plasma transaminase concentrations and cellular injury in general, indicated by elevated plasma LDH levels. The exaggerated proinflammatory and injurious response in A2AP−/− mice most likely explains their accelerated mortality.
It remains to be determined whether the observed effects of A2AP deficiency during infection with B. pseudomallei also apply to infections with other gram-negative bacteria. Published studies from our group indicate that PAI-1 (like A2AP, an inhibitor of fibrinolysis) has similar protective roles in pneumonia-derived sepsis caused by either B. pseudomallei (22) or Klebsiella pneumoniae (21). Although these previous data cannot be directly extrapolated to A2AP−/− mice, they suggest that the current results may also be applicable to other gram-negative pathogens. Clearly, independent experiments are required to establish this.
Caution is needed when extrapolating data from mouse experiments to human disease. Murine models like the one used here make use of a homogenous group of experimental animals with identical genotype, sex, and (relatively young) age exposed to a well-controlled bacterial challenge, whereas patients form a heterogeneous group in which multiple factors modify disease outcome, including extent of pathogen exposure, older age, comorbidities, comedication, and genetic composition. Taking these precautions into mind, the present study is the first to describe the possible protective role of A2AP in a clinically relevant model of severe gram-negative sepsis.
The authors thank M. ten Brink and J. Daalhuisen for their expert technical assistance during the animal experiments; G. C. K. W. Koh and T. J. Hommes for their assistance in processing the experimental samples; R. de Beer for performing histopathological and immunohistochemical stainings; S. Havik and Dr. H. R. Lijnen for breeding the A2AP−/− mice; J. W. Duitman for performing the aTGF-β1 measurements; W. F. Kopatz, M. A. Weijne, and L. M. Leverink for performing the coagulation measurements; and A. J. Hoogendijk for his assistance in formatting the image files.
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Supported by The Netherlands Organization for Health Research and Development (ZonMW) grants 92003504 (L.M.K.) and 90700424 (W.J.W.), the Stichting BeGeTu (L.M.K.), Dutch Kidney Foundation grant C09.2287 (J.J.T.H.R.), and The Netherlands Organization for Scientific Research (NWO) VENI grant 91610008 (W.J.W.).
Author Contributions: L.M.K. performed the study; collected, analyzed and interpreted the data; and wrote the first draft of the article. T.A.W., W.J.W., J.J.T.H.R., J.C.M.M., and A.M.D. assisted in collection and interpretation of the data. C.v.V. assisted in interpretation of the data. T.v.d.P. designed the study and assisted in interpretation of the data. All authors contributed in revising the manuscript and finally approved the version of the manuscript to be published.
This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.201307-1344OC on August 30, 2013
Author disclosures are available with the text of this article at www.atsjournals.org.
