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, 13 (10), e0205521
eCollection

CXCL4 Contributes to Host Defense Against Acute Pseudomonas Aeruginosa Lung Infection

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CXCL4 Contributes to Host Defense Against Acute Pseudomonas Aeruginosa Lung Infection

Lei Yue et al. PLoS One.

Abstract

Platelets have been implicated in pulmonary inflammation following exposure to bacterial stimuli. The mechanisms involved in the platelet-mediated host response to respiratory bacterial infection remain incompletely understood. In this study, we demonstrate that platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) plays critical roles in a mouse model of acute bacterial pneumonia using Pseudomonas aeruginosa. Platelets are activated during P. aeruginosa infection, and mice depleted of platelets display markedly increased mortality and impaired bacterial clearance. CXCL4 deficiency impairs bacterial clearance and lung epithelial permeability, which correlate with decreased neutrophil recruitment to BALF. Interestingly, CXCL4 deficiency selectively regulates chemokine production, suggesting that CXCL4 has an impact on other chemokine expression. In addition, CXCL4 deficiency reduces platelet-neutrophil interactions in blood following P. aeruginosa infection. Further studies revealed that platelet-derived CXCL4 contributes to the P. aeruginosa-killing of neutrophils. Altogether, these findings demonstrate that CXCL4 is a vital chemokine that plays critical roles in bacterial clearance during P. aeruginosa infection through recruiting neutrophils to the lungs and intracellular bacterial killing.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Platelet depletion impairs host defense against acute P. aeruginosa pulmonary infection in mice.
(A, B, C, D) Platelets were activated after P. aeruginosa lung infection. Wild-type mice were intranasally infected with 1×109 CFU of P. aeruginosa strain 8821 or an equivalent volume of saline (NT). Blood and BALF were collected 24 hpi. Platelet activation in the blood and BALF was assessed via flow cytometry (n = 4 ± SEM, *p < 0.05, ***p < 0.001). (E) Platelet depletion decreases animal survival following P. aeruginosa infection. Sixteen hours prior to infection with P. aeruginosa, wild-type mice were administered an intraperitoneal injection of 50 μL of rabbit anti-mouse platelet serum. Wild-type and platelet-depleted mice were intranasally infected with saline (NT) or 1×109 CFU of P. aeruginosa strain 8821. Animal survival was monitored for 7 days post infection (n = 10 ± SEM). (F) Platelet depletion impairs bacterial clearance following P. aeruginosa lung infection. Wild-type and platelet-depleted mice were intranasally infected with 1×109 CFU of P. aeruginosa strain 8821 for 24 h. Lungs were collected at 24 hpi. Serial dilutions of homogenized lung tissues were streaked onto LB agar plates and incubated for 24 h at 37°C. The resulting colonies were counted to determine the bacterial load (n = 9 ± SEM, *p < 0.05). (G, H) CXCL4 is mainly activated in the location that P. aeruginosa infected. Wild-type mice were intranasally infected with 1×109 CFU of P. aeruginosa strain 8821 for 24 h. Serum and BALF supernatants were collected for determining CXCL4 production (n = 3 ± SEM, **p < 0.01, ***p < 0.001).
Fig 2
Fig 2. Chemokine (C-X-C motif) ligand 4 (CXCL4) deficiency impairs bacterial clearance and airway epithelial permeability after P. aeruginosa lung infection.
(A, B, C, D) CXCL4 deficiency results in impaired bacterial clearance following P. aeruginosa lung infection but it does not affect host mortality and morbidity. Wild-type and CXCL4-/- mice were intranasally infected with 1×109 CFU of P. aeruginosa strain 8821 for 24 h. BALF and lungs were collected at 24 hpi. Serial dilutions of homogenized BALF and lung tissue were streaked onto LB agar plates and incubated 24 h at 37°C. The resulting colonies were counted to determine bacteria load (n = 9 ± SEM, *p < 0.05). For the survival study, wild-type and CXCL4-/- mice were intranasally infected with 1×109 CFU of P. aeruginosa strain 8821. Animal survival and body weight were monitored up to 7 days post infection (n = 12 ± SEM). (E, F) CXCL4-deficient mice display increased airway epithelial permeability after P. aeruginosa infection. Wild-type and CXCL4-/- mice were left uninfected (NT) or intranasally infected with 1×109 CFU of P. aeruginosa strain 8821. Six hours after infection, the mice received intraperitoneal injections of Evans blue dye. Blood, lungs and BALF were collected at 24 hpi. Dye leakage into the BALF and lungs was assessed and is presented as the OD620 ratio to a 1:20 dilution of serum (n = 4 ± SEM, **p < 0.01).
Fig 3
Fig 3. CXCL4 deficiency impairs neutrophil recruitment with regulating production of LIX after P. aeruginosa lung infection.
(A, B) Wild-type and CXCL4-/- mice were intranasally infected with 1×109 CFU of P. aeruginosa strain 8821 or an equivalent volume of saline (NT). BALF and lungs were collected 24 hpi. Neutrophil recruitment to the BALF were assessed by MPO activity (n = 9 ± SEM, **p < 0.01) and Flow Cytometry (n = 4 ± SEM, *p < 0.05). (C-D) Supernatants of the BALF and lung homogenate were analyzed for production of LIX (n = 8 ± SEM, *p < 0.05, **p < 0.01).
Fig 4
Fig 4. CXCL4 deficiency reduces platelet-neutrophil interactions without affecting platelet or neutrophil number in blood following P. aeruginosa lung infection.
Wild-type and CXCL4-/- mice were intranasally infected with 1×109 CFU of P. aeruginosa strain 8821 or an equivalent volume of saline (NT). Blood were collected at 24 hpi. Red blood cells were lysed with lysis buffer. The cells were stained with Ly6G (neutrophils) and CD41 (platelets). The Ly6G+ CD41+ cell population was gated as illustrated (A, B) and the platelets interacted with neutrophils (C, D) was analyzed by flow cytometry. The total numbers of platelet and neutrophil were also counted (E, F) (n = 4 ± SEM, **p < 0.01).
Fig 5
Fig 5. CXCL4 contributes to the P. aeruginosa-killing capability of neutrophils.
(A, B) The CXCL4 levels in neutrophils and platelets was evaluated after P. aeruginosa infection in vitro. Wild-type bone marrow-isolated neutrophils and blood isolated-platelets were left untreated (NT) or exposed to P. aeruginosa strain 8821 at a MOI of 1:10 for 6 h. Supernatants were subjected to ELISA analysis for determining CXCL4 production (n = 3 ± SEM, **p < 0.01, ***p < 0.001). (C) The phagocytosis index was significantly decreased in CXCL4-deficient neutrophil. Mouse bone marrow-derived neutrophils were isolated from WT or CXCL4-/- mice, and then incubated with P. aeruginosa 8821 (opsonized with mouse serum) at 37°C for 30 min. The cells were prepared using Cytospin. The specimens were then stained with Diff-Quik and examined under a microscope. The number of bacteria engulfed by 100 randomly selected neutrophils was counted. The phagocytic activity was measured according to the phagocytosis index (n = 3 ± SEM, *p<0.05). (D) Bacterial killing capacity was impaired in CXCL4-deficient neutrophil. Mouse bone marrow-derived neutrophils were isolated from WT or CXCL4-/- mice and incubated with P. aeruginosa 8821 (opsonized with mouse serum) at 37°C for 1 h. Gentamycin was added to kill extracellular bacteria for 3 h. Then, the neutrophils were washed and lysed with PBS containing 0.1% Triton X-100. The samples were serially diluted and spread onto Luria broth (LB) agar plates. Colony numbers were determined after overnight incubation at 37°C (n = 3 ± SEM, **p<0.01). (E, F) Wild-type and CXCL4-/- bone marrow isolated neutrophil or blood isolated platelet were left untreated (NT) or exposed to P. aeruginosa strain 8821 at a MOI of 1:10. Supernatants were collected at 6h and analyzed for NO production (n = 3 ± SEM, ***p < 0.001). (G, H) platelet-derived CXCL4 may facilitate the neutrophil-mediated clearance of P. aeruginosa. Mouse bone marrow-derived neutrophils and blood-derived platelets were isolated from WT or CXCL4-deficient mice. An in vitro phagocytosis assay and intracellular killing assay were performed as described above. (n = 3 ± SEM, *p<0.05).

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References

    1. Cripps AW, Dunkley ML, Clancy RL, Kyd J. Pulmonary immunity to Pseudomonas aeruginosa. Immunology and cell biology. 1995;73(5):418–24. 10.1038/icb.1995.65 . - DOI - PubMed
    1. Speert DP. Phagocytosis of Pseudomonas aeruginosa by macrophages: receptor-ligand interactions. Trends in microbiology. 1993;1(6):217–21. . - PubMed
    1. Wesselkamper SC, Eppert BL, Motz GT, Lau GW, Hassett DJ, Borchers MT. NKG2D is critical for NK cell activation in host defense against Pseudomonas aeruginosa respiratory infection. Journal of immunology. 2008;181(8):5481–9. ; PubMed Central PMCID: PMC2567053. - PMC - PubMed
    1. Powderly WG, Pier GB, Markham RB. T lymphocyte-mediated protection against Pseudomonas aeruginosa infection in granulocytopenic mice. The Journal of clinical investigation. 1986;78(2):375–80. 10.1172/JCI112587 ; PubMed Central PMCID: PMC423557. - DOI - PMC - PubMed
    1. Ritchie AJ, Yam AO, Tanabe KM, Rice SA, Cooley MA. Modification of in vivo and in vitro T- and B-cell-mediated immune responses by the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone. Infection and immunity. 2003;71(8):4421–31. 10.1128/IAI.71.8.4421-4431.2003 ; PubMed Central PMCID: PMC165988. - DOI - PMC - PubMed

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Grant support

TJL has received grant support from the National Natural Science Foundation of China (81471564) (http://www.nsfc.gov.cn). LY is supported by Yunnan Natural Science Foundation (2016FB037, 2017FB019) (http://www.ynstc.gov.cn), PUMC Youth Fund (3332016114) (http://www.cams.ac.cn), Fundamental Research Funds for the Central Universities (2016ZX350070) (http://www.cams.ac.cn) and CAMS Innovation Fund for Medical Sciences (2017-I2M-2-006) (http://www.cams.ac.cn). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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