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Extracellular Adenosine Protects against Streptococcus pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment.

Bou Ghanem EN, Clark S, Roggensack SE, McIver SR, Alcaide P, Haydon PG, Leong JM - PLoS Pathog. (2015)

Bottom Line: We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden.Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs.The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to S. pneumoniae lung infection. Moreover, adenosine diminished PMN movement across endothelial monolayers in vitro, and although inhibition or deficiency of CD73 had no discernible impact on PMN recruitment within the first 6 hours after intratracheal inoculation of mice, these measures enhanced PMN numbers in the pulmonary interstitium after 18 hours of infection, culminating in dramatically elevated numbers of pulmonary PMNs at three days post-infection. When assessed at this time point, CD73-/- mice displayed increased levels of cellular factors that promote leukocyte migration, such as CXCL2 chemokine in the murine lung, as well as CXCR2 and β-2 integrin on the surface of pulmonary PMNs. The enhanced pneumococcal susceptibility of CD73-/- mice was significantly reversed by PMN depletion following infection, suggesting that EAD-mediated resistance is largely mediated by its effects on PMNs. Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs. The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.

No MeSH data available.


Related in: MedlinePlus

PMNs promote pulmonary and systemic disease during later stages of S. pneumoniae lung infection.(A) C57BL/6J mice were inoculated I.T with 5x105 CFU of S. pneumoniae TIGR4 and pulmonary (green) and bloodstream (red) bacterial loads, as well as pulmonary PMNs (blue) were monitored through 72 hours post-infection. Shown are representative data from one of two separate experiments (using 3 to 4 mice per time point). The numbers above the graph represent the fraction of surviving mice within that group at the corresponding time. (B-D) C57BL/6J mice were treated i.p with PMN depleting antibodies (anti-ly6G) or isotype control either 18 hours pre or post pulmonary challenge with 5x105 CFU of S. pneumoniae TIGR4. Survival (B) and bacterial burdens in the blood (C) were monitored over time and shown are pooled data from two separate experiments. (D) Pneumococcal burdens in the lungs and blood were determined 3 days post-infection. The numbers below the graph represent the fraction of surviving mice within that group. Representative data from one of 4 separate experiments with 3 to 4 mice per group are shown. Means +/- SEM are given in Panels A, C and D, and values significantly (p<0.05) different from isotype control-treated group by Student’s t-test are indicated by asterisk. In Panel B, asterisk indicates survival rate was significantly (p<0.05) different from isotype control-treated controls by Log-rank (Mantel-Cox) test.
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ppat.1005126.g001: PMNs promote pulmonary and systemic disease during later stages of S. pneumoniae lung infection.(A) C57BL/6J mice were inoculated I.T with 5x105 CFU of S. pneumoniae TIGR4 and pulmonary (green) and bloodstream (red) bacterial loads, as well as pulmonary PMNs (blue) were monitored through 72 hours post-infection. Shown are representative data from one of two separate experiments (using 3 to 4 mice per time point). The numbers above the graph represent the fraction of surviving mice within that group at the corresponding time. (B-D) C57BL/6J mice were treated i.p with PMN depleting antibodies (anti-ly6G) or isotype control either 18 hours pre or post pulmonary challenge with 5x105 CFU of S. pneumoniae TIGR4. Survival (B) and bacterial burdens in the blood (C) were monitored over time and shown are pooled data from two separate experiments. (D) Pneumococcal burdens in the lungs and blood were determined 3 days post-infection. The numbers below the graph represent the fraction of surviving mice within that group. Representative data from one of 4 separate experiments with 3 to 4 mice per group are shown. Means +/- SEM are given in Panels A, C and D, and values significantly (p<0.05) different from isotype control-treated group by Student’s t-test are indicated by asterisk. In Panel B, asterisk indicates survival rate was significantly (p<0.05) different from isotype control-treated controls by Log-rank (Mantel-Cox) test.

Mentions: To better understand the role of PMNs following pneumococcal infection, C57BL/6 mice were infected intra-tracheally (I.T.) with 5x105 colony-forming units (CFU) of S. pneumoniae TIGR4 strain and pulmonary PMN influx and bacterial burdens in the lungs and blood were monitored for 72 hours. The total number of pulmonary PMNs, determined by flow cytometric analysis of a single-cell suspension of whole lung, increased four-fold in the first three hours post-infection, then underwent a dramatic increase, peaking at 30 million, i.e. ~100-fold greater than uninfected controls, at 18 hours post-infection (Fig 1A). Between 24 and 72 hours post-infection, as mice started to succumb to the disease, surviving mice experienced an ~10-fold decrease in pulmonary PMNs (Fig 1A).


Extracellular Adenosine Protects against Streptococcus pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment.

Bou Ghanem EN, Clark S, Roggensack SE, McIver SR, Alcaide P, Haydon PG, Leong JM - PLoS Pathog. (2015)

PMNs promote pulmonary and systemic disease during later stages of S. pneumoniae lung infection.(A) C57BL/6J mice were inoculated I.T with 5x105 CFU of S. pneumoniae TIGR4 and pulmonary (green) and bloodstream (red) bacterial loads, as well as pulmonary PMNs (blue) were monitored through 72 hours post-infection. Shown are representative data from one of two separate experiments (using 3 to 4 mice per time point). The numbers above the graph represent the fraction of surviving mice within that group at the corresponding time. (B-D) C57BL/6J mice were treated i.p with PMN depleting antibodies (anti-ly6G) or isotype control either 18 hours pre or post pulmonary challenge with 5x105 CFU of S. pneumoniae TIGR4. Survival (B) and bacterial burdens in the blood (C) were monitored over time and shown are pooled data from two separate experiments. (D) Pneumococcal burdens in the lungs and blood were determined 3 days post-infection. The numbers below the graph represent the fraction of surviving mice within that group. Representative data from one of 4 separate experiments with 3 to 4 mice per group are shown. Means +/- SEM are given in Panels A, C and D, and values significantly (p<0.05) different from isotype control-treated group by Student’s t-test are indicated by asterisk. In Panel B, asterisk indicates survival rate was significantly (p<0.05) different from isotype control-treated controls by Log-rank (Mantel-Cox) test.
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ppat.1005126.g001: PMNs promote pulmonary and systemic disease during later stages of S. pneumoniae lung infection.(A) C57BL/6J mice were inoculated I.T with 5x105 CFU of S. pneumoniae TIGR4 and pulmonary (green) and bloodstream (red) bacterial loads, as well as pulmonary PMNs (blue) were monitored through 72 hours post-infection. Shown are representative data from one of two separate experiments (using 3 to 4 mice per time point). The numbers above the graph represent the fraction of surviving mice within that group at the corresponding time. (B-D) C57BL/6J mice were treated i.p with PMN depleting antibodies (anti-ly6G) or isotype control either 18 hours pre or post pulmonary challenge with 5x105 CFU of S. pneumoniae TIGR4. Survival (B) and bacterial burdens in the blood (C) were monitored over time and shown are pooled data from two separate experiments. (D) Pneumococcal burdens in the lungs and blood were determined 3 days post-infection. The numbers below the graph represent the fraction of surviving mice within that group. Representative data from one of 4 separate experiments with 3 to 4 mice per group are shown. Means +/- SEM are given in Panels A, C and D, and values significantly (p<0.05) different from isotype control-treated group by Student’s t-test are indicated by asterisk. In Panel B, asterisk indicates survival rate was significantly (p<0.05) different from isotype control-treated controls by Log-rank (Mantel-Cox) test.
Mentions: To better understand the role of PMNs following pneumococcal infection, C57BL/6 mice were infected intra-tracheally (I.T.) with 5x105 colony-forming units (CFU) of S. pneumoniae TIGR4 strain and pulmonary PMN influx and bacterial burdens in the lungs and blood were monitored for 72 hours. The total number of pulmonary PMNs, determined by flow cytometric analysis of a single-cell suspension of whole lung, increased four-fold in the first three hours post-infection, then underwent a dramatic increase, peaking at 30 million, i.e. ~100-fold greater than uninfected controls, at 18 hours post-infection (Fig 1A). Between 24 and 72 hours post-infection, as mice started to succumb to the disease, surviving mice experienced an ~10-fold decrease in pulmonary PMNs (Fig 1A).

Bottom Line: We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden.Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs.The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to S. pneumoniae lung infection. Moreover, adenosine diminished PMN movement across endothelial monolayers in vitro, and although inhibition or deficiency of CD73 had no discernible impact on PMN recruitment within the first 6 hours after intratracheal inoculation of mice, these measures enhanced PMN numbers in the pulmonary interstitium after 18 hours of infection, culminating in dramatically elevated numbers of pulmonary PMNs at three days post-infection. When assessed at this time point, CD73-/- mice displayed increased levels of cellular factors that promote leukocyte migration, such as CXCL2 chemokine in the murine lung, as well as CXCR2 and β-2 integrin on the surface of pulmonary PMNs. The enhanced pneumococcal susceptibility of CD73-/- mice was significantly reversed by PMN depletion following infection, suggesting that EAD-mediated resistance is largely mediated by its effects on PMNs. Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs. The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.

No MeSH data available.


Related in: MedlinePlus