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Efferocytosis impairs pulmonary macrophage and lung antibacterial function via PGE2/EP2 signaling.

Medeiros AI, Serezani CH, Lee SP, Peters-Golden M - J. Exp. Med. (2009)

Bottom Line: Moreover, intrapulmonary administration of ACs demonstrated that PGE(2) generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo.These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses.They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE(2) or ligation of EP2.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health Systems, Ann Arbor, MI 48109, USA.

ABSTRACT
The ingestion of apoptotic cells (ACs; termed "efferocytosis") by phagocytes has been shown to trigger the release of molecules such as transforming growth factor beta, interleukin-10 (IL-10), nitric oxide, and prostaglandin E(2) (PGE(2)). Although the antiinflammatory actions of these mediators may contribute to the restoration of homeostasis after tissue injury, their potential impact on antibacterial defense is unknown. The lung is highly susceptible to diverse forms of injury, and secondary bacterial infections after injury are of enormous clinical importance. We show that ACs suppress in vitro phagocytosis and bacterial killing by alveolar macrophages and that this is mediated by a cyclooxygenase-PGE(2)-E prostanoid receptor 2 (EP2)-adenylyl cyclase-cyclic AMP pathway. Moreover, intrapulmonary administration of ACs demonstrated that PGE(2) generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo. These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses. They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE(2) or ligation of EP2.

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Intrapulmonary administration of ACs impairs host defense in a mouse model of pneumococcal pneumonia. (A) Thymocytes were incubated with 1 μM dexamethasone for 6 h and ACs were detected by AnnexinV-FITC/PI and analyzed by flow cytometry. Early ACs comprise 40.3% of total cells. (B) 106 CFU of S. pneumoniae and varying numbers of apoptotic thymocytes were coadministered intratracheally in WT mice. Lung homogenates were assessed for bacterial CFUs 48 h later. (C) Indicated numbers of apoptotic or viable thymocytes were instilled intranasally in WT mice and, 16 h later, 106 CFU S. pneumoniae were administered intratracheally. Lung homogenates were assessed for bacterial CFUs 48 h after S. pneumoniae challenge. (D) Bacterial CFUs were determined in blood obtained 48 h after S. pneumoniae challenge from the same WT mice studied in C. (E) WT and EP2−/− mice were subjected to intranasal administration of apoptotic thymocytes 16 h before intratracheal challenge with S. pneumoniae as described in C. Lung homogenate CFUs 48 h after bacterial challenge are presented. (F) Bacterial CFUs were determined in blood obtained 48 h after S. pneumoniae challenge from the same EP2−/− mice studied in E. Results represent the mean ± SEM of one experiment representative of two. The number of animals analyzed in each group is indicated above each bar. ND, none detected. *, P < 0.05 versus control; #, P < 0.05 versus AC.
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fig4: Intrapulmonary administration of ACs impairs host defense in a mouse model of pneumococcal pneumonia. (A) Thymocytes were incubated with 1 μM dexamethasone for 6 h and ACs were detected by AnnexinV-FITC/PI and analyzed by flow cytometry. Early ACs comprise 40.3% of total cells. (B) 106 CFU of S. pneumoniae and varying numbers of apoptotic thymocytes were coadministered intratracheally in WT mice. Lung homogenates were assessed for bacterial CFUs 48 h later. (C) Indicated numbers of apoptotic or viable thymocytes were instilled intranasally in WT mice and, 16 h later, 106 CFU S. pneumoniae were administered intratracheally. Lung homogenates were assessed for bacterial CFUs 48 h after S. pneumoniae challenge. (D) Bacterial CFUs were determined in blood obtained 48 h after S. pneumoniae challenge from the same WT mice studied in C. (E) WT and EP2−/− mice were subjected to intranasal administration of apoptotic thymocytes 16 h before intratracheal challenge with S. pneumoniae as described in C. Lung homogenate CFUs 48 h after bacterial challenge are presented. (F) Bacterial CFUs were determined in blood obtained 48 h after S. pneumoniae challenge from the same EP2−/− mice studied in E. Results represent the mean ± SEM of one experiment representative of two. The number of animals analyzed in each group is indicated above each bar. ND, none detected. *, P < 0.05 versus control; #, P < 0.05 versus AC.

Mentions: To confirm the biological significance of these in vitro results, we modeled a secondary lung infection after initial pulmonary exposure to ACs such as what would be seen in acute lung injury. For these experiments, ACs were generated by dexamethasone treatment of murine thymocytes for 6 h, which yielded 40.3% early apoptotic and 4.93% late apoptotic plus necrotic cells (Fig. 4 A). Initially, different numbers of ACs were coadministered intratracheally in C57BL/6 WT mice along with a standard inoculum of the important respiratory pathogen Streptococcus pneumoniae, and 48 h after challenge the bacterial burdens in lung homogenates were evaluated. As shown in Fig. 4 B, there was no difference in pulmonary bacterial clearance of mice that were infected and simultaneously exposed to ACs using this protocol compared with those infected alone. Because our in vitro results indicated that the efferocytosis-induced inhibition of FcR-mediated phagocytosis was time dependent and reached a level of 89% when phagocytic target challenge was performed after an interval of 16 h (Fig. 1 C), we devised a second model in which S. pneumoniae was administered intratracheally 16 h after various numbers of apoptotic thymocytes were instilled intranasally. As ∼55% of the total numbers of cells obtained after treatment of thymocytes with dexamethasone remain viable, viable cells were also administrated intranasally as an experimental control. Results showed that pretreatment with ACs, but not viable cells, using this protocol dose-dependently impaired pulmonary bacterial clearance (Fig. 4 C) and also led to the dissemination of S. pneumoniae into the bloodstream (Fig. 4 D) 48 h after infection. Finally, to test the role of the EP2 receptor in this impairment of in vivo pulmonary defense against S. pneumoniae by AC pretreatment, we compared the lung and the bloodstream bacterial burdens in WT versus EP2−/− mice. In contrast with WT mice, the pulmonary bacterial burden in AC-pretreated EP2−/− mice was no greater than in non–AC-pretreated controls but was 2.5 logs lower than in AC-pretreated WT mice (Fig. 4 E). In addition, EP2−/− mice exhibited no bacteremia (Fig. 4 F). Because these mice lack preexisting antibodies against S. pneumoniae, bacterial recognition and clearance by phagocytes in the in vivo model is likely independent of FcR, indicating that the PGE2/EP2/cAMP axis also suppresses innate immune responses when bacterial recognition proceeds via other relevant recognition receptors such as toll-like receptors (19), collectins (20), or scavenger receptors (21).


Efferocytosis impairs pulmonary macrophage and lung antibacterial function via PGE2/EP2 signaling.

Medeiros AI, Serezani CH, Lee SP, Peters-Golden M - J. Exp. Med. (2009)

Intrapulmonary administration of ACs impairs host defense in a mouse model of pneumococcal pneumonia. (A) Thymocytes were incubated with 1 μM dexamethasone for 6 h and ACs were detected by AnnexinV-FITC/PI and analyzed by flow cytometry. Early ACs comprise 40.3% of total cells. (B) 106 CFU of S. pneumoniae and varying numbers of apoptotic thymocytes were coadministered intratracheally in WT mice. Lung homogenates were assessed for bacterial CFUs 48 h later. (C) Indicated numbers of apoptotic or viable thymocytes were instilled intranasally in WT mice and, 16 h later, 106 CFU S. pneumoniae were administered intratracheally. Lung homogenates were assessed for bacterial CFUs 48 h after S. pneumoniae challenge. (D) Bacterial CFUs were determined in blood obtained 48 h after S. pneumoniae challenge from the same WT mice studied in C. (E) WT and EP2−/− mice were subjected to intranasal administration of apoptotic thymocytes 16 h before intratracheal challenge with S. pneumoniae as described in C. Lung homogenate CFUs 48 h after bacterial challenge are presented. (F) Bacterial CFUs were determined in blood obtained 48 h after S. pneumoniae challenge from the same EP2−/− mice studied in E. Results represent the mean ± SEM of one experiment representative of two. The number of animals analyzed in each group is indicated above each bar. ND, none detected. *, P < 0.05 versus control; #, P < 0.05 versus AC.
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fig4: Intrapulmonary administration of ACs impairs host defense in a mouse model of pneumococcal pneumonia. (A) Thymocytes were incubated with 1 μM dexamethasone for 6 h and ACs were detected by AnnexinV-FITC/PI and analyzed by flow cytometry. Early ACs comprise 40.3% of total cells. (B) 106 CFU of S. pneumoniae and varying numbers of apoptotic thymocytes were coadministered intratracheally in WT mice. Lung homogenates were assessed for bacterial CFUs 48 h later. (C) Indicated numbers of apoptotic or viable thymocytes were instilled intranasally in WT mice and, 16 h later, 106 CFU S. pneumoniae were administered intratracheally. Lung homogenates were assessed for bacterial CFUs 48 h after S. pneumoniae challenge. (D) Bacterial CFUs were determined in blood obtained 48 h after S. pneumoniae challenge from the same WT mice studied in C. (E) WT and EP2−/− mice were subjected to intranasal administration of apoptotic thymocytes 16 h before intratracheal challenge with S. pneumoniae as described in C. Lung homogenate CFUs 48 h after bacterial challenge are presented. (F) Bacterial CFUs were determined in blood obtained 48 h after S. pneumoniae challenge from the same EP2−/− mice studied in E. Results represent the mean ± SEM of one experiment representative of two. The number of animals analyzed in each group is indicated above each bar. ND, none detected. *, P < 0.05 versus control; #, P < 0.05 versus AC.
Mentions: To confirm the biological significance of these in vitro results, we modeled a secondary lung infection after initial pulmonary exposure to ACs such as what would be seen in acute lung injury. For these experiments, ACs were generated by dexamethasone treatment of murine thymocytes for 6 h, which yielded 40.3% early apoptotic and 4.93% late apoptotic plus necrotic cells (Fig. 4 A). Initially, different numbers of ACs were coadministered intratracheally in C57BL/6 WT mice along with a standard inoculum of the important respiratory pathogen Streptococcus pneumoniae, and 48 h after challenge the bacterial burdens in lung homogenates were evaluated. As shown in Fig. 4 B, there was no difference in pulmonary bacterial clearance of mice that were infected and simultaneously exposed to ACs using this protocol compared with those infected alone. Because our in vitro results indicated that the efferocytosis-induced inhibition of FcR-mediated phagocytosis was time dependent and reached a level of 89% when phagocytic target challenge was performed after an interval of 16 h (Fig. 1 C), we devised a second model in which S. pneumoniae was administered intratracheally 16 h after various numbers of apoptotic thymocytes were instilled intranasally. As ∼55% of the total numbers of cells obtained after treatment of thymocytes with dexamethasone remain viable, viable cells were also administrated intranasally as an experimental control. Results showed that pretreatment with ACs, but not viable cells, using this protocol dose-dependently impaired pulmonary bacterial clearance (Fig. 4 C) and also led to the dissemination of S. pneumoniae into the bloodstream (Fig. 4 D) 48 h after infection. Finally, to test the role of the EP2 receptor in this impairment of in vivo pulmonary defense against S. pneumoniae by AC pretreatment, we compared the lung and the bloodstream bacterial burdens in WT versus EP2−/− mice. In contrast with WT mice, the pulmonary bacterial burden in AC-pretreated EP2−/− mice was no greater than in non–AC-pretreated controls but was 2.5 logs lower than in AC-pretreated WT mice (Fig. 4 E). In addition, EP2−/− mice exhibited no bacteremia (Fig. 4 F). Because these mice lack preexisting antibodies against S. pneumoniae, bacterial recognition and clearance by phagocytes in the in vivo model is likely independent of FcR, indicating that the PGE2/EP2/cAMP axis also suppresses innate immune responses when bacterial recognition proceeds via other relevant recognition receptors such as toll-like receptors (19), collectins (20), or scavenger receptors (21).

Bottom Line: Moreover, intrapulmonary administration of ACs demonstrated that PGE(2) generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo.These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses.They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE(2) or ligation of EP2.

View Article: PubMed Central - PubMed

Affiliation: Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health Systems, Ann Arbor, MI 48109, USA.

ABSTRACT
The ingestion of apoptotic cells (ACs; termed "efferocytosis") by phagocytes has been shown to trigger the release of molecules such as transforming growth factor beta, interleukin-10 (IL-10), nitric oxide, and prostaglandin E(2) (PGE(2)). Although the antiinflammatory actions of these mediators may contribute to the restoration of homeostasis after tissue injury, their potential impact on antibacterial defense is unknown. The lung is highly susceptible to diverse forms of injury, and secondary bacterial infections after injury are of enormous clinical importance. We show that ACs suppress in vitro phagocytosis and bacterial killing by alveolar macrophages and that this is mediated by a cyclooxygenase-PGE(2)-E prostanoid receptor 2 (EP2)-adenylyl cyclase-cyclic AMP pathway. Moreover, intrapulmonary administration of ACs demonstrated that PGE(2) generated during efferocytosis and acting via EP2 accounts for subsequent impairment of lung recruitment of polymorphonuclear leukocytes and clearance of Streptococcus pneumoniae, as well as enhanced generation of IL-10 in vivo. These results suggest that in addition to their beneficial homeostatic influence, antiinflammatory programs activated by efferocytosis in the lung have the undesirable potential to dampen innate antimicrobial responses. They also identify an opportunity to reduce the incidence and severity of pneumonia in the setting of lung injury by pharmacologically targeting synthesis of PGE(2) or ligation of EP2.

Show MeSH
Related in: MedlinePlus