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The tissue factor pathway in pneumonia
van den Boogaard, F.E.
Publication date
2015
Document Version
Final published version
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Citation for published version (APA):
van den Boogaard, F. E. (2015). The tissue factor pathway in pneumonia.
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PART III
PLATELETS IN
PNEUMOCOCCAL PNEUMONIA
CHAPTER 10
Thrombocytopenia impairs host defense
during murine Streptococcus pneumoniae
pneumonia
Florry E. van den Boogaard MD1, 2, Marcel Schouten MD, PhD1,2,Sacha F. de Stoppelaar MD1,2, Joris J.T.H. Roelofs MD, PhD3,
Xanthe Brands1,2, Marcus J. Schultz MD, PhD4,5,
Cornelis van ’t Veer PhD1,2, Tom van der Poll MD, PhD1,2,6
Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands:1Center for Experimental and Molecular Medicine (CEMM),2Center for Infection and Immunity Amsterdam (CINIMA),3Department of Pathology,4Department of Intensive Care Medicine,5Laboratory of Experimental Intensive Care and Anesthesiology (LEICA),6Division of Infectious Diseases
ABSTRACT
Objective: Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. In patients, thrombocytopenia is correlated with an adverse outcome of pneumonia. Platelets can modulate the host response to infection in several ways, i.e. by facilitating clot formation, production of antimicrobial proteins and interaction with neutrophils. We studied the effect of thrombocytopenia during murine pneumococcal pneumonia.
Design: Animal study.
Setting: University research laboratory. Subjects: Mice.
Interventions: Pneumonia was induced by intranasal inoculation of S. pneumoniae. Platelets were depleted by anti-mouse thrombocyte serum; controls received
non-immunogenic serum. In separate studies mice were treated with the platelet P2Y12
receptor inhibitor clopidogrel or placebo.
Measurements and Main Results: Thrombocytopenic mice (platelet counts <1% of unin-fected controls) showed a reduced survival during pneumococcal pneumonia (27% ver-sus 75% amongst controls; p=0.003), which was associated with higher bacterial loads in lungs, spleen, and blood. Thrombocytopenic mice showed enhanced coagulation ac-tivation (thrombin-antithrombin complexes) in plasma. Proinflammatory cytokine levels were higher in plasma but not in lungs of thrombocytopenic mice. Although clopidogrel treatment strongly prolonged the bleeding time, it did not impact on bacterial loads during pneumococcal pneumonia.
Conclusions: Platelets play a protective role during pneumococcal pneumonia indepen-dent of their aggregation.
205 Thrombocytopenia in pneumococcal pneumonia
10
INTRODUCTION
Streptococcus (S.) pneumoniae is the most prevalent microorganism in
community-acquired pneumonia (CAP) and an important causative organism in sepsis in this
con-text1, 2. In the United States alone, the pneumococcus is responsible for more than half a
million CAP cases each year and 50,000 episodes of bacteremia with a case fatality rate
of 7 and 20% respectively3. Similar figures have been reported for Europe4 and stress the
importance of expanding our knowledge of the host defense mechanisms that influ-ence the outcome of pneumococcal pneumonia.
Thrombocytopenia is a common finding in patients admitted to the Intensive Care
Unit (ICU) and associated with a worse outcome5-7. Moreover, in a clinical study including
over 800 ICU patients with CAP, thrombocytopenia was an independent predictor of
mortality8. Platelets are well known chief cellular effectors of hemostasis, maintaining
vascular integrity at sites of injury and inflammation. More recent investigations have revealed that platelets contribute to diverse processes that extend beyond hemostasis and thrombosis. A compelling body of evidence now exists indicating that platelets also
contribute to inflammatory processes and defense against infection9-11.
Platelets act on the host innate and adaptive inflammatory response via release of a wide variety of preformed peptides stored in granules that mediate inflammation,
chemotaxis and wound repair9-11. They are able to interact directly with neutrophils and
this interaction can be induced by plasma from septic patients9, 12. During systemic13-15
and lung inflammation16, 17 platelets sequestrate in lungs, which in experimental models
was neutrophil dependent13, 16, 18; conversely, platelet depletion inhibited neutrophil
recruitment during acute lung injury in mice18-20. In addition, preventing
neutrophil-platelet interaction protected from lung tissue damage during lung injury16, 18, 20-22.
Through expression of pattern recognition receptors13, 23 platelets also interact directly
and indirectly with pathogens; they can bind, aggregate and internalize microorgan-isms24, 25 and can augment antibacterial activities of other immune cells26-28. Additional
antibacterial effects are achieved by production of antimicrobial mediators (e.g. cyto-toxic oxygen metabolites) or platelet microbicidal proteins known as “thrombocidins” in
humans, of which increased levels are found during infection11, 29, 30. Recent studies also
demonstrated that platelets are able to induce neutrophil extracellular trap formation to
entrap bacteria in septic blood12.
Clearly platelets are equipped to play an important role in host defense against invad-ing pathogens and this contribution may be especially important in the lung where they are found to accumulate during an inflammatory assault. We here aimed to study the role of platelets during CAP, for which we depleted platelets or inhibited platelet aggre-gation and secondary activation in mice prior to intranasal infection with S. pneumoniae.
MATERIALS AND METHODS Animals
Specific pathogen-free C57BL/6 mice were purchasedfrom Charles River (Maastricht,
The Netherlands). Experiments were conducted with age and gender-matched mice at
10–12 weeks of age. The Institutional Animal Careand Use Committee ofthe Academic
Medical Centerapproved all experiments.
Experimental study design
S. pneumoniae serotype 3 (American Type Culture Collection, ATCC 6303, Rockville, MD)
was used to induce pneumococcal pneumonia. Bacteria were grown as described31
and ~5 x 104 colony-forming units (CFU) in 50 µL were inoculated intranasally. Mice
were observed in a survival experiment or sacrificed at 24 or 48 hours after induction of pneumococcal pneumonia. Per group eight animals were used, for the survival experiment 15 animals per group were used. In order to deplete platelets, mice were intravenously treated with rabbit anti-mouse platelet serum or normal rabbit serum as control (Accurate Chemical & Scientific Corporation, Westbury, NY) 2 hours before
and at 24 and 48 hours after induction of pneumonia13. Treatment effect was verified by
determining platelet counts in a counting chamber. In one experiment, platelets were depleted using rat anti-mouse platelet glycoprotein Ib alpha chain (GP1b-α) 50 µg per
mouse intravenously32, 33; controls received nonimmune rat IgG (both antibodies from
Emfret Analytics GmbH & Co, Würzburg, Germany). In separate experiments, mice were treated with the platelet aggregation and activation inhibitor clopidogrel (30 mg/kg, Mylan, Hoeilaart, Belgium) or placebo by oral gavage at day -2, -1 and at time of
infec-tion as used previously34. Clopidogrel is an irreversible inhibitor of P2Y
12, the receptor
for adenosine diphosphate on platelets35. Blood diluted 4:1 with citrate, lungs, liver and
spleen were harvested and processed using methods described previously31. The left
lung lobe was fixed in 10% buffered formalin and embedded in paraffin. Bacterial quantification
Undiluted whole blood and serial 10–fold dilutions of organ homogenates and whole blood were made in sterile isotonic saline and plated onto sheep–blood agar plates. Colony forming units (CFU) were counted following 16 hours of incubation at 37°C. Assays
Thrombin-antithrombin complexes (TATc) were measured using a commercially avail-able enzyme-linked immunosorbent assay (ELISA) (TATc: Behringwerke AG, Marburg, Germany, or Affinity Biologicals, Ancaster, Ontario, Canada), levels of macrophage–in-flammatory protein (MIP)-2, keratinocyte-derived cytokine (KC), interleukin (IL)-1β
207 Thrombocytopenia in pneumococcal pneumonia
10
(all R&D Systems, Abingdon, UK) and myeloperoxidase (MPO; HyCult Biotechnology, Uden, The Netherlands) were measured using commercially available ELISA kits. Levels of tumor necrosis factor (TNF)-α, IL–6, monocyte chemotactic protein (MCP)–1 and interferon–gamma (IFN–γ) were determined using a commercially available cytometric beads array multiplex assay (BD Biosciences, San Jose, CA) in accordance with the manu-facturers’ recommendations.
Tail vein bleeding assay
A mouse-tail vein bleeding assay was performed as described (27). In brief, at a standard-ized distance, the tip of the mouse-tail was cut off (with a tail diameter approximately 1 mm). The tail was immediately placed in a 50-mL falcon tube filled with 37 °C saline and the bleeding time with a maximum of 5 minutes was recorded.
Histopathology and immunohistochemistry
Paraffin-embedded 4-μm-thick lung sections were stained with hematoxylin and eosin (H&E) and analyzed for hemorrhage, inflammation and tissue damage, as described
previously31, 36. All slides were coded and scored by a pathologist who was blinded for
group identity. Hemorrhage was scored separately and total pathology scores were de-termined as the sum of the following variables: interstitial inflammation, endothelialitis, bronchitis, edema, pleuritis and presence of thrombi. Confluent (diffuse) inflammatory infiltrate was quantified separately and expressed as percentage of total lung surface. The remaining variables were rated on a scale from 0 (condition absent) to 4 (most severe condition). Neutrophil stainings on mouse lung tissue were performed using an anti-mouse Ly-6G monoclonal antibody (BD Pharmingen, San Diego, CA). Slides were photographed with a microscope equipped with a digital camera (Olympus dotSlide,
Tokyo, Japan) and stained areas were analyzed with ImageJ (version 2006.02.01; US
National Institutes of Health) and expressed as percentage of the total lung surface area
as described elsewhere37.
Statistical analysis
Data are expressed as indicated. Differences between groups were analyzedusing
non-parametric analysis of variance (rank transformed variables) with modeled effects for strain and time, followed by post-hoc Mann-Whitney U tests at the individual time points. Survival curves and bleeding time were compared using log-rank test. All analyses were done using GraphPad Prism (GraphPad Software, San Diego, CA, USA). A p-value of less
RESULTS
Thrombocytopenic mice show enhanced lethality in S. pneumoniae pneumonia To study the effect of thrombocytopenia on mortality in an experimental model of CAP
we depleted platelets in mice using anti-mouse platelet serum13. Before infection with S.
pneumoniae, platelet counts were reduced to less than 1% (p = 0.03) and after infection
to less than 10% in mice treated with rabbit anti-mouse platelet serum compared to mice treated with control rabbit serum (p = 0.002, Figure 1A). Thrombocytopenic mice showed a strongly enhanced mortality (73%) compared with control mice (25%, p = 0.003, Figure 1B) after induction of S. pneumoniae pneumonia.
Thrombocytopenia is associated with higher systemic bacterial loads during pneumococcal pneumonia
To evaluate the effect of thrombocytopenia on bacterial loads during pneumococcal pneumonia, bacteria were quantified locally (lung homogenates) and systemically (whole blood and spleen homogenates). Thrombocytopenic mice demonstrated a trend toward higher bacterial counts in lungs at 24 hours (p = 0.065, Figure 2A) together with increased dissemination, as reflected by higher bacterial loads in blood at both time points (p < 0.05, Figure 2B) and spleen at 24 hours (p < 0.001, Figure 2C). To confirm that thrombocytopenia facilitates bacterial dissemination, we depleted platelets in mice by
a different approach, using an antibody directed against GP1b-α32, 33. In accordance with
previous studies32, 33, anti-GP1b-α treatment reduced platelet counts to 7 ± 2% relative
hours 0 50 100 150 0 20 40 60 80 100 C um ul at ie ve s ur vi va l control anti-P serum ** A. B. 0 500 1000 1500 2000 24h 48h control anti-P serum 10 ^6 th ro m bo cy te s/ m L uninfected * *** ***
Figure 1. Thrombocytopenia enhances lethality in pneumococcal pneumonia.
Mice were treated with rabbit anti-mouse platelet (P) serum or control serum and infected intranasally with Streptococcus pneumoniae. Platelet counts in mice treated with anti-mouse platelet (anti-P) or control serum at 0, 24 or 48 hours after infection (A). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (n = 4 per uninfected group and n = 8 per infected group). *p < 0.05 and ***p <0.001 compared with control mice. Platelet counts of uninfected thrombocytopenic mice at 0, 24 and 48 hours are 11.46 ± 4.6, 47.2 ± 15.5, and 81.4 ± 13.7 x 106/mL (mean ± SEM) respectively. Cumulative survival of control (closed symbols) and thrombocytopenic (open symbols) mice (n = 15 per group) (B) **p < 0.01 compared to controls, log rank test.
209 Thrombocytopenia in pneumococcal pneumonia
10
to controls (p = 0.03). Similar to mice made thrombocytopenic with antiplatelet serum, anti-GP1b-α-treated mice displayed increased bacterial loads in blood and spleen (Fig 2E, F) at 24 hours after infection with S. pneumoniae via the airways (both p < 0.05).
Thrombocytopenia does not impact on lung inflammation during pneumococcal pneumonia
Platelets have been found to contribute to several forms of sterile lung inflammation at
least in part by facilitating neutrophil recruitment9, 16, 19. We were therefore interested in
the impact of thrombocytopenia on the pulmonary inflammatory response to infection with S. pneumoniae. The extent of lung pathology, as determined by the semi-quan-titative histology scores described in the Materials and Methods section, did not differ between thrombocytopenic and control mice (Figure 3G, with representative slides for control A-C and thrombocytopenic mice D-F 0, 24 and 48 hours after infection). Notably, signs of pulmonary hemorrhage were almost exclusively found in thrombocytopenic mice in the setting of infection (Figure 3H); thrombocytopenia did not result in hemor-rhage in uninfected mice. Thrombocytopenia did not influence neutrophil influx during pneumonia, as indicated by the number of Ly6+ cells in lung tissue slides and concentra-tions of MPO in whole-lung homogenates (Figure 4). Similarly, thrombocytopenia did
Spleen Lung Blood Blood Spleen Lung 0 1 2 3 4 5 6 7 8 9 10 10lo g C FU /m L IgG a-GP1b-0 1 2 3 4 5 6 10lo g C FU /m L * 0 1 2 3 4 5 6 7 10lo g C FU /m L * A. B. C. D. E. F. 0 1 2 3 4 5 6 10lo g C FU /m L ** 24h 48h * 0 1 2 3 4 5 6 7 10lo gC FU /m l *** 24h 48h 0 1 2 3 4 5 6 7 8 9 10 10lo gC FU /m l 24h 48h control anti-P serum
Figure 2. Thrombocytopenia is associated with higher systemic bacterial loads in pneumococcal pneumonia. Mice were depleted of platelets and infected intranasally with Streptococcus pneumoniae.
Number of colony-forming units (CFUs) per milliliter lung homogenates (A, D), whole blood (B, E) and spleen homogenates (C, F) 24 and 48 hours after infection in mice treated with rabbit anti-platelet (P) serum (open boxes) or control serum (grey boxes) (A-C) and in mice treated with rat anti-mouse platelet glycopro-tein Ib alpha chain (a-GP1b-α) (striped boxes) or nonimmune rat IgG (grey boxes) 24 hours after infection (D-F). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (n = 8 per group). *p < 0.05 and **p < 0.01 compared with controls.
not affect cytokine or chemokine levels in lung homogenates, with the exception of MCP-1 (48h) concentrations, which were higher in lungs of thrombocytopenic mice (Table 1). Altogether these data indicate that thrombocytopenia did not have a major impact on the local inflammatory response during pneumococcal pneumonia.
Thrombocytopenia enhances the systemic cytokine response during pneumococcal pneumonia
To obtain insight in the systemic inflammatory response, we measured cytokine and chemokine levels in plasma harvested 24 or 48 hours after infection. At 24 hours after infection plasma levels of these mediators did not differ between groups; at 48 hours
control anti-P serum 0h 24h0h 0h 0 2 4 6 8 10 12 not detected 48h 24h 0h control anti-P serum to ta l P A sc or e 0 1 2 3 4 5 # 48h 24h ** 0h not detected bl ee di ng score 48h control anti-P serum 200µm 200µm 200µm 200µm 200µm 200µm A. B. C. D. E. F. G. H.
Figure 3. Thrombocytopenia does not impact on lung histopathology, but is associated with intra-pulmonary hemorrhage during pneumococcal pneumonia. Mice were depleted of platelets and
in-fected intranasally with Streptococcus pneumoniae. Lung tissue samples were obtained at 0, 24 or 48 hours postinfection. Representative microphotographs of haematoxylin and eosin-stained lung sections of mice treated with control (A-C) or anti-platelet (P) serum (D-F) at 0, 24 and 48 hours, and total lung histopathol-ogy (PA) (G) and bleeding (H) scores of control (grey boxes) and thrombocytopenic (open boxes) mice.
Ar-rows indicate sites of hemorrhage. Data are expressed as box-and-whisker diagrams depicting the smallest
observation, lower quartile, median, upper quartile and largest observation (n = 8 per group). Scale bar indicates 200 μm. # p < 0.10 and ** p < 0.01 compared with controls.
211 Thrombocytopenia in pneumococcal pneumonia
10
after infection, however, thrombocytopenic mice showed strongly increased plasma concentrations of TNF-α, IL-6, IFN-γ and MCP-1 (Table 2).
Thrombocytopenia is associated with an enhanced procoagulant response during pneumococcal pneumonia
Platelets are important for hemostasis and amplification of thrombin generation38. To
evaluate the effect of thrombocytopenia on coagulation in mice, we measured TATc levels in plasma before and 24 and 48 hours after infection (Figure 5). Directly before infection, TATc levels were similar in thrombocytopenic and control mice. At 24 and 48 hours postinfection, thrombocytopenia was associated with increased plasma levels of TATc (p = 0.06) and p < 0.001 respectively).
0 10000 20000 30000 40000 M PO [p g/ m l] control anti-P serum 0 5 10 15 Ly 6G score (%) 48h 24h A. B. D. C. 0h 24h 48h control anti-P serum
control anti-P serum
Figure 4. Thrombocytopenia does not influence neutrophil accumulation in lung tissue during pneu-mococcal pneumonia. Mice were treated with rabbit antiplatelet (P) serum or control serum and infected
intranasally with Streptococcus pneumoniae; samples were obtained at 0, 24 or 48 hours postinfection. Con-centrations of myeloperoxidase (MPO) per milliliter whole-lung homogenate (A) and neutrophil accumula-tion in lung tissue expressed as total Ly-6G positivity as percentage of lung tissue surface (B) in control (grey boxes) and thrombocytopenic (open boxes) mice with representative microphotographs of Ly-6G staining of control (C) and thrombocytopenic (D) mice at 48 hours. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (n = 8 per group). Scale bar indicates 200 μm.
Clopidogrel does not influence bacterial loads or the inflammatory response during pneumococcal pneumonia
To investigate whether the protective role of platelets in pneumococcal pneumonia
depends on the aggregation and secondary activation of platelets, we inhibited P2Y12
receptor-mediated aggregation and activation of platelets using clopidogrel34. Although
this treatment was effective as reflected by a profoundly increased tail vein bleeding time (p = 0.007) (Figure 6A), it did not affect bacterial counts in any of the organs tested at either 24 or 48 hours postinfection (Figure 6B-E). Of note, no macroscopic or intra-pulmonary signs of bleeding were found in mice treated with clopidogrel. In addition,
Table 1. Cytokine and chemokine concentrations in lung homogenates
24 hr 48 hr Control (n=8) Anti-platelet (n=8) Control (n=8) Anti-platelet (n=8) TNFα (pg/ml) 114 (19-432) 714 (194-1094) 380 (74-441) 305 (167-548) IL-6 (pg/ml) 433 (71-2993) 1870 (173-2512) 2994 (941-3459) 3339 (1597-4097) MCP-1 (pg/ml) 1753 (720-4534) 5329 (1706-6490) 5931 (3401-8034) 9789 (8136-10240)* IFNγ (pg/ml) 11 (3-50) 19 (5-42) 59 (17-84) 62 (31-149) IL-10 (pg/ml) 132 (119-218) 170 (106-331) 71 (45-89) 105 (69-216) KC (pg/ml) 4581 (1266-17224) 10295 (3989-13991) 39806 (26774-52093) 40814 (25092-44103) MIP2 (pg/ml) 2342 (1916-5207) 6342 (3109-19318) 45126 (9848-56297) 19276 (9490-39503) IL-1β (pg/ml) 568 (72-1393) 1666 (402-3919) 870 (452-979) 439 (294-617)
Levels of cytokines and chemokines in lung homogenates 24 and 48 hr after induction of pneumococ-cal pneumonia in mice treated with anti-mouse platelet serum (anti-platelet) or control serum. Data are expressed as median (interquartile ranges). IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; KC, keratinocyte-derived cytokine; MCP-1, monocyte chemotactic protein-1; MIP-2, Macrophage–inflamma-tory protein–2. * p < 0.05 versus control.
Table 2. Cytokine and chemokine levels in plasma
24 hr 48 hr control (n=8) anti-platelet (n=7) control (n=8) anti-platelet (n=8) TNFα (pg/ml) 12 (9-24) 28 (11-32) 19 (9-25) 55 (32-82)*** IL-6 (pg/ml) 67 (10-238) 253 (2-329) 128 (82-166) 416 (214-620)** MCP-1 (pg/ml) 131 (67-522) 415 (113-987) 308 (254-428) 1070 (818-2011)*** IFNγ (pg/ml) 7 (2-47) 32 (3-80) 11 (8-14) 293 (148-977)*** IL-12 (pg/ml) 28 (5-39) 34 (5-64) 9 (4-19) 24 (10-57)
Levels of cytokines and chemokines in plasma 24 and 48 hr after induction of pneumococcal pneumonia in mice treated with anti-mouse platelet (antiplatelet) serum or control serum. Data are expressed as median (interquartile range). IL, interleukin; IFN, interferon; TNF, tumor necrosis factor; MCP-1, monocyte chemo-tactic protein-1. **and *** indicate p < 0.01 and p < 0.001 versus control.
213 Thrombocytopenia in pneumococcal pneumonia
10
control clopidogrel 0 60 120 180 240 300 Ta il bl ee di ng ti m e (s ec ) ** upper limit 0 2 4 6 8 10 10lo g C FU /m l control clopidogrel 24h 48h 0 1 2 3 4 5 6 7 8 10lo g C FU /m l n.d. 24h 48h 1 2 3 4 5 6 7 8 10lo g C FU /m l 24h 48h 1 2 3 4 5 6 7 8 10lo g C FU /m l 24h 48h A. B. C. E. D. control clopidogrel control clopidogrel control clopidogrel Lung Liver Spleen Blood b.d. b.d.Figure 6. Inhibition of thrombocyte activation by clopidogrel increases bleeding time but does not influence bacterial loads during pneumococcal pneumonia. Mice were infected intranasally with Strep-tococcus pneumoniae and treated with the platelet activation inhibitor clopidogrel or placebo; samples
were harvested 24 or 48 hours postinfection. Tail vein bleeding time (A) and number of colony-forming units (CFUs) per milliliter lung (B), liver (C), spleen (D) homogenates and whole blood (E) in control mice (grey boxes) and mice treated with clopidogrel (striped boxes). Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observa-tion (n = 8 per group). ** p < 0.01 compared with controls. n.d = not determined; b.d = below detecobserva-tion.
0 5 10 15 20 TA Tc [n g/ m l] control anti-P serum #
***
uninfected 24h 48hFigure 5. Thrombocytopenia is associated with an enhanced procoagulant response during pneu-mococcal pneumonia. Mice were treated with anti-platelet (P) serum or control serum and infected
in-tranasally with Streptococcus pneumoniae. Thrombin-antithrombin complex (TATc) levels in plasma of un-infected or un-infected control (grey boxes) and thrombocytopenic (open boxes) mice at 24 and 48 hours postinfection. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile and largest observation (n = 8 per group). *** p < 0.001, # p < 0.1 compared with controls.
clopidogrel did not influence lung pathology or the systemic cytokine response (data not shown).
DISCUSSION
Besides their well-known hemostatic function, platelets are increasingly appreciated as key effectors in inflammatory responses. Thrombocytopenia frequently occurs in criti-cally ill patients, and lower platelet counts are an independent risk factor of mortality in
patients admitted to the ICU for severe CAP8. We here investigated the role of platelets
during CAP using thrombocytopenic mice infected with S. pneumoniae via the airways and show that thrombocytopenia facilitates bacterial dissemination, resulting in en-hanced systemic inflammation and increased mortality.
The absence of platelets promoted bacterial dissemination, indicating that platelets contribute to local containment of pneumococcal infection in the lung. Likewise, in rat endocarditis caused by S. sanguinis, the bacterial density of the valve vegetations
dose-dependently increased with platelet depletion39. In addition, platelets promote
endo-thelial barrier integrity9, 40 and in experimental animals thrombocytopenia increased
pulmonary vascular permeability41; however, to date it is not clear whether platelets also
influence the lung epithelial barrier. In contrast to our results, in a recent study, platelets
facilitated dissemination of Streptococcus pyogenes in murine sepsis42. This implies that
distinct streptococcal species vary in their interaction with platelets. Indeed, S. pyogenes can rapidly adhere to and promote aggregation of platelets, while S. pneumoniae
ap-pears to optimally induce aggregation by an antibody-dependent mechanism11, 43.
Platelets can modulate the immune response in several ways9. In experimental models
of sterile acute lung injury, thrombocytopenia protected against pulmonary injury16, 20,
whereas it worsened lung injury in septic shock44. We found little effect on lung damage
caused by platelet depletion after induction of pneumococcal pneumonia, as reflected by similar lung histopathology scores and pulmonary cytokine and chemokine levels. Of note, pneumonia caused major lung hemorrhage during thrombocytopenia, in line with
reports from previous studies using noninfectious challenges33, 45, again implicating
platelets are required to maintain vascular barrier integrity in the setting of inflamma-tion. In a recent study demonstrating a protective effect of platelets during septic shock, no lung hemorrhage was observed and it was suggested that this was due to a milder extent of platelet depletion, that is 90% compared to more than 97.5% in the
aforemen-tioned studies44. In the studies carried out here, platelets were depleted for ~90% 48
hours after pneumococcal infection, still major hemorrhage was observed, which could be attributed to the fact that the site of primary infection was located in the lungs.
215 Thrombocytopenia in pneumococcal pneumonia
10
Platelet depletion inhibited neutrophil recruitment into lungs in several studies of
acute lung injury in mice18-20, 22, 46. However, in an other study of murine endotoxemia,
platelet depletion did not affect neutrophil accumulation13. In line with the latter study,
we did not observe any difference in neutrophil counts in lung tissue between control and thrombocytopenic mice, as reflected by similar numbers of Ly6-G positive cells and MPO levels in lungs. At the same time, we demonstrated higher cytokine levels in plasma of thrombocytopenic mice during pneumococcal pneumonia, which may at least in part be explained by the higher number of bacteria present in the circulation. Furthermore, it was recently shown that platelets inhibit IL-6 and TNF-α production in endotoxemia
in a macrophage dependent manner44. Therefore, a direct effect of platelet depletion
on cytokine release may have contributed to the increased systemic cytokine response observed here. An overwhelming, ongoing systemic inflammatory response likely has added to tissue damage and mortality in thrombocytopenic mice.
Next to their central role in primary hemostasis, activated platelets are able to induce a coagulation response as they facilitate coagulation by providing a suitable
phos-pholipid surface for the assembly of activated coagulation factors47. Surprisingly, we
found enhanced activation of coagulation in plasma of thrombocytopenic mice during infection, which together with enhanced systemic inflammation may have resulted in increased microthrombotic organ failure. This, combined with major bleeding complica-tions associated with strongly reduced platelet counts, likely contributed to enhanced lethality in thrombocytopenic mice. The increased coagulation response may, in part, be attributed to an augmented systemic proinflammatory response in thrombocytopenic
mice. In addition, S. pneumoniae is able to activate coagulation48 and increased bacterial
counts in plasma of thrombocytopenic mice may provide an alternative explanation for the enhanced procoagulant state. Together, these results clearly show that platelets are not required for coagulation activation during pneumococcal pneumonia.
Activation of platelets is a typical feature during systemic inflammation49 and S.
pneu-moniae can induce platelet aggregation50. Retrospective data investigating the outcome
patients with CAP suggested a benefit for those patients who had used antiplatelet
drugs for over 6 months before hospital admission51. We studied the effect of platelet
aggregation and secondary activation inhibition with clopidogrel during pneumococ-cal pneumonia. Clopidogrel markedly increased the bleeding time in mice, but did not impact on bacterial loads or dissemination. Together these data indicate that the mere inhibition of the hemostatic properties of platelets does not impact on host defense in the setting of pneumonia caused by S. pneumoniae, whereas decreasing platelet numbers does.
In conclusion, we demonstrated here that platelets, independent of aggregation and secondary activation, play a favorable role in the setting of experimental CAP caused by
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SUPPLEMENTARY MATERIAL
Supplementary Table 1. Platelet counts in uninfected, platelet depleted mice.
0 hr 24 hr 48 hr
11,46 ± 4,6 x 106 47,2 ± 15,5 x 106 81,4 ± 13,7 x 106
Platelet counts x 106/mL in uninfected mice treated at 0, 24 and 48 hours (hr) after treatment with anti-platelet serum.
