Hypersusceptibility to Invasive Pneumococcal Infection in Experimental Sickle Cell Disease Involves Platelet-Activating Factor Receptor
- 1Department of Infectious Diseases, Comprehensive Sickle Cell Center, St. Jude Children's Research Hospital, Memphis Tennessee
- 2Division of Experimental Hematology, Comprehensive Sickle Cell Center, St. Jude Children's Research Hospital, Memphis Tennessee
- Reprints or correspondence: Elaine I. Tuomanen, Dept. of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105 (elaine.tuomanen{at}stjude.org).
Abstract
Children with sickle cell disease have a 600-fold increased incidence of invasive pneumococcal disease. Platelet-activating factor receptor (PAFr) mediates pneumococcal invasion, and up-regulation of PAFr on chronically activated endothelia could contribute to increased bacterial invasion. Mice transplanted with sickle cell bone marrow developed more extensive infection, and 57% died, compared with 16% of wild-type mice. Histopathological analysis revealed that sickle cell mice expressed significantly more PAFr on endothelia and epithelia. Pharmacological blockade or genetic deletion of PAFr protected sickle cell mice from mortality. We conclude that PAFr plays an important role in hypersusceptibility to pneumococcal infection in sickle cell disease.
Streptococcus pneumoniae is the leading cause of serious bacterial infection in the pediatric population and is estimated to cause 17,000 cases of bacteremia each year [1]. Children with sickle cell disease have 400–600 times greater risk of invasive infection with pneumococcus than their healthy peers, and the presentation of disease in these patients is often a fulminant, lethal sepsis [1]. This invasive disease rate is well beyond the 2–3-fold increased risk of sepsis from other encapsulated bacteria.
Insight into the mechanism underlying the increased vulnerability of children with sickle cell disease to invasive pneumococcal infection could lead to more effective disease prevention. Epidemiological studies have suggested that the rate of carriage of pneumococci in sickle cell patients is equivalent to healthy children (30%–40%) [2], indicating that the difference in incidence of invasive disease may relate to events after colonization. No defects in amounts of complement components, properdin, or neutrophil-killing capability have been found [3]. Splenic function decreases progressively in sickle cell disease and may compromise bacterial clearance. Lower concentrations of opsonizing anticapsular antibody have also been suggested to contribute to sepsis [4, 5]. However, decreased antibody levels or decreased splenic function would be expected to cause an increase in incidence of invasive disease due to all encapsulated pathogens of childhood, not just the pneumococcus. Yet, the heightened risk is not shared by other encapsulated pathogens or by other anemias [1], suggesting that there is a potentially causal relationship between severity of pneumococcal disease and the sickle cell phenotype.
As a consequence of chronic hypoxia and infection, patients with sickle cell disease exhibit a state of perpetual endothelial activation [6–8]. The increased expression of adhesion molecules and carbohydrate receptors exhibited on the sickle cell endothelium supports this notion. Platelet-activating factor receptor (PAFr), a G protein-coupled receptor that binds the lipid chemokine PAF, is widely expressed throughout the body and is up-regulated by inflammation [9]. The critical role that PAFr plays in pneumococcal invasion has been clearly established in vitro and in vivo [10–13]. Phosphorylcholine on the pneumococcal cell wall mimics the bioactive component of PAF and serves to insert the bacteria into the PAFr uptake pathway to enter human cells. Bacterial translocation across the vascular endothelium is severely impaired in pafr−/− mice and in wild-type mice treated with PAFr antagonists [10]. Using a murine model of sickle cell disease, hypersusceptibility to pneumococcal infection was recapitulated, and we tested the hypothesis that PAFr is up-regulated in sickle cell disease and promotes development of severe pneumococcal infection.
Materials and methods. Lethally irradiated C57B/6J mice were transplanted as described [14–16] with 2×106 to 3 × 106 bone marrow cells from either BERK sickle cell mice (n = 23), cells from mice with β-thalassemia (n=24) [17], or cells from wild-type mice (n=31). β-thalassemic mice were used as a control for anemia. Baytril (enrofloxacin; 2.27% solution, diluted 1:100 in drinking water; Bayer) was administered as an antimicrobial prophylaxis for 3–4 weeks after transplantation. To assess the impact that PAFr has on survival, pafr−/− mice (n=4) [10] were transplanted with BERK sickle cell bone marrow. In addition to Baytril, these mice required supportive weekly red blood cell transfusions for 5 weeks after transplantation. After a 3–4 month engraftment period, full engraftment with each donor marrow was confirmed by hemoglobin cellulose acetate electrophoresis of red cell lysates [18].
Mice were challenged intranasally (inl) with 1×107 to 2 × 107 cfu/mouse of serotype 2 Streptococcus pneumoniae strains D39 or bioluminescent D39X [19]. In some cases (n=4), mice were treated with PAFr antagonist BN52021 (200 µg/mouse intraperitoneally; BIOMOL) 1 h before and daily after challenge. Mice were imaged with the IVIS Xenogen imaging system (Xenogen) to characterize the spread of bacteria. Kaplan-Meier analysis and generalized Wilcoxon and Mann-Whitney U tests were performed (StatsDirect software; version 2.5.7) to determine statistical significance of mortality and differences in bacterial load. To compare the intensity of PAFr staining, liver, lung, and spleen samples were obtained from mice perfused with 0.1 mol/L cacodylic acid solution with 3% paraformaldehyde and stained with anti-PAFr antibody (Santa Cruz Biotechnology) and the Vectastain Elite ABC Kit (Vector Lab-oratories).
Results and discussion. Inl challenge most closely approximates natural acquisition of pneumococci in the nasopharynx of humans followed by progression to pneumonia and bacteremia. Sickle cell, β-thalassemia, and wild-type mice were challenged inl with pneumococci. At 24 h, Xenogen imaging revealed pneumonia was prominent in wild-type and β-thalassemic mice, whereas sickle cell mice had already progressed rapidly to sepsis (figure 1A). At 48 h, median bacterial loads in blood were generally 2 logs higher in the sickle cell mice, although the ranges of bacterial counts were wide: 4×106 cfu/mL (95% confidence interval [CI], 2.8×105 to 2.9×108 cfu/mL) for wild-type mice (n=15) and 1×108 cfu/mL (95% CI, 1.5×107 to 2.3×109 cfu/mL) for sickle cell mice (n=3) (P=.154). Mean survival times were 51 h (95% CI, 42–60 h) for sickle cell mice, 62 h (95% CI, 53–71 h) for β-thalassemia mice, and 66 h (95% CI, 61–72 h) for wild-type mice. Fifty-seven percent of sickle cell mice expired by 48 h after infection, compared with 21% for β-thalassemia and 16% for wild-type mice (figure 1B). Death rates were significantly different between the sickle cell and wild-type groups (P=.023), as well as between sickle cell and β-thalassemia groups (P=.014) but not between β-thalssemia and wild-type groups (P=.105). Thus, as with humans, sickle cell mice experienced a significantly more aggressive course of disease than wild-type mice, and this was apparently not due to anemia per se.
Figure 1
Course of pneumococcal infection in wild-type (WT) versus sickle cell (SS) mice.A, Bioluminescent pneumococci were visualized in living mice at 24 h. Bacteria were localized to the lung in WT mice, whereas SS mice were diffusely infected and bacteremic. The amount of bacteria is proportional to the intensity of color, with blue being low and red to yellow being high titer. B, Kaplan-Meier analysis of survival of SS versus WT mice challenged with pneumococci. WT (n=31), SS (n=23), and β-thalassemic (βthal; n=24) mice were challenged intranasally with pneumococci and followed for mortality for 72 h. SS vs. WT, P=.023; SS vs. βthal, P=.014; βthal vs. WT, P=.105.
PAFr is the eukaryotic receptor facilitating pneumococcal translocation across endothelia and epithelia. Because PAFr is up-regulated by inflammation, we hypothesized that sickle cell mice would display more PAFr and blockade of the receptor would attenuate the course of infection. Figure 2A shows an increased intensity of staining for PAFr at the airway epithelium and vascular endothelium of the lung in sickle cell mice, compared with that of wild-type mice both before and after infection. Sections of liver and spleen also showed greater PAFr staining in sickle cell than in wild-type mice even in the absence of infection (data not shown), demonstrating that contact with bacteria does not explain the activated sickle cell endothelium displaying more PAFr.
Figure 2
Involvement of platelet-activating factor receptor (PAFr) in pneumococcal disease in sickle cell (SS) mice. A, Immunohistochemical staining for PAFr, performed on lung samples from wild-type (WT) and SS mice before and 18 h after bacterial challenge. Anti-PAFr antibody binding was detected with horseradish peroxidase-conjugated secondary antibody (brown). B, Improvement in survival of SS mice with intervention at PAFr. Untreated SS mice (n=10; black bar), SS pafr−/− mice (n=4; white bar), and SS mice treated with PAFr antagonist (n=4; gray bar) were challenged with pneumococci and followed for survival. *Significantly different (P<.05). Br, bronchus; V, vessel.
The impact that blockade of PAFr has on the survival of sickle cell mice was evaluated by challenge of sickle cell pafr−/− mice (n=4) and by administration of PAFr antagonist to sickle cell mice wild type for PAFr (n=4) (figure 2B). At 24 h, none of the pafr−/− sickle cell mice or PAFr antagonist-treated sickle cell mice died, whereas 30% of the untreated sickle cell mice died. This improvement approached the level of protection in control mice transplanted with normal bone marrow (P=.011). Comparison of the groups at 48 h showed continued improved survival in the pafr−/− mice, whereas the antagonist benefit was lost (figure 2B). Thus, complete absence of PAFr afforded sustained protection from pneumococcal-induced mortality, whereas pharmacological intervention with PAFr was transiently protective.
As invasive infection involves intimate bacterial contact with and penetration of the vascular wall, activated endothelium is likely to participate in the course of sepsis. We have shown that the resting endovasculature and pulmonary epithelium of mice with sickle cell disease is sufficiently activated so as to display enhanced PAFr that facilitates an aggressive, invasive course of pneumococcal infection. Absence of PAFr or receptor blockade attenuates infection and represents a potential for therapeutic intervention to decrease the severity of invasive pneumococcal disease in patients with sickle cell disease.
Footnotes
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Potential conflicts of interest: none reported.
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Financial support: National Institutes of Health (grants AI 27913 and AI 39482); National Heart, Lung, and Blood Institute (grant U54HL070590); American Lebanese Syrian Associated Charities.
- Received July 5, 2006.
- Accepted September 20, 2006.
- © 2007 by the Infectious Diseases Society of America
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