Adhesion of Streptococcus pneumoniae to human airway epithelial cells exposed to urban particulate matter Naseem Mushtaq, PhD, Majid Ezzati, PhD, Lucinda.

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Presentation transcript:

Adhesion of Streptococcus pneumoniae to human airway epithelial cells exposed to urban particulate matter Naseem Mushtaq, PhD, Majid Ezzati, PhD, Lucinda Hall, PhD, Iain Dickson, BSc, Michael Kirwan, PhD, Ken M.Y. Png, PhD, Ian S. Mudway, PhD, Jonathan Grigg, MD, FRCPCH Journal of Allergy and Clinical Immunology Volume 127, Issue 5, Pages 1236-1242.e2 (May 2011) DOI: 10.1016/j.jaci.2010.11.039 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Effect of urban PM10 (UK) on the adhesion of S pneumoniae D39 to A549 cells. PM10 dose was indexed to ultrafine carbon black by using the optical absorbance of Brown et al.21 Cells were cultured with PM10 for 4 hours, then infected with S pneumoniae for 2 hours. Adherence of bacteria was determined by quantitative culture and is expressed as CFU count/mL. Incubation with PM10 (UK) at 50 μg/mL increases CFU count (∗P < .001 by t test). Data are represented as means and SEMs (n ≥ 5) and are representative of >3 separate experiments. Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Adhesion of D39 S pneumoniae to A549 cells assessed by confocal microscopy. A549 cells were stained by Hoescht stain, then cultured with medium or PM10 (UK) 50 μg/mL, then cultured with S pneumoniae (n = 5 for each data point). Culture of cells with PM10 (UK) increases mean S pneumoniae fluorescence. Data are represented as means and SEMs (n = 50 cells) and are representative of 3 separate experiments. ∗P < .01 by t test. Left panel shows a representative image of A549 cells cultured in medium. Adherent S pneumoniae are red. Right panel shows cells cultured with 50 μg/mL PM10 (UK) then cultured with S pneumoniae. Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 A549 cells cultured with PM10 (UK) 50 μg/mL and treated with antibiotics to kill cell-surface S pneumoniae leaving only intracellular bacteria. CFU was determined by quantitative culture. Incubation of cells with PM10 (UK) increases the intracellular CFU count. ∗P < .01 versus medium control by t test. Data are represented as means and SEMs (n ≥ 5) and are representative of >3 experiments. Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Adhesion of D39 S pneumoniae to human primary bronchial epithelial cells (HBEpC) cultured with 50 μg/mL PM10 (UK). PM10 increases pneumococcal adhesion in primary cells. Basal and stimulated adhesion in primary cells is lower than A549 cells. ∗P < .05, ∗∗P < .01 versus medium control by t test. Data are represented as means and SEMs (n ≥ 5) and are representative of >3 separate experiments. Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Effect of urban PM10 and PM2.5 (Ghana) on the adhesion of S pneumoniae to A549 cells. Urban PM10 and PM2.5 (Ghana) increases pneumococcal adhesion. ∗P < .05, ∗∗P < .01 versus medium control by t test. PM (Ghana)–stimulated adhesion is lower than PM10 (UK)–stimulated adhesion (P < .05). Data are represented as means and SEMs (n ≥ 5) and are representative of 3 separate experiments. Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 A, Transmission electron microscopy (TEM) image of an A549 cell cultured with PM10 (UK). Black electron-dense material represents PM. Bar = 2 μm, ×7000. B, TEM of an A549 cell under higher magnification showing PM10 (UK) both within intracellular vesicles and free in the cytoplasm. Bar = 2 μm, ×10,000. C, Scanning electron microcopy image of A549 cells after culture with PM10 (UK) and S pneumoniae. The white irregular surface material is PM, and adherent pneumococci are beadlike structures in cell-surface depressions (arrow). Bar = 50 μm, ×1200. Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Effect of the antioxidant NAC (30 mmol/mL) on PM10 (UK)–stimulated adhesion of S pneumoniae to A549 cells. The number of adherent bacteria was determined by quantitative culture and expressed as CFU/mL. Incubation of A549 cells with N-acetyl cysteine (NAC) before infection partly attenuates the PM10 (UK)–stimulated increase in CFU count. ∗P <.05 versus PM10 without NAC by t test. NAC has no significant effect on CFU count in medium control cells (P = NS versus cells without NAC). Data are shown as means and SEMs (n = 5) and are representative of 3 separate experiments. Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 8 A, PAFR transcript level in A549 cells cultured with PM10 (UK) 50 μg/mL for 4 hours. Transcript level was assessed by quantitative RT-PCR and normalized for the housekeeping gene GAPDH. The relative change in expression was calculated by using the comparative Ct method, and data expressed as % of control. Data show means and SEMs (n = 5 in triplicate wells). PM10 (UK) increased PAFR transcript level. ∗P < .01 versus control by t test. B, PAFR expression on A549 cells assessed by confocal microscopy. A549 cells were cultured with PM10 (UK) 50 μg/mL for 4 hours. Mean fluorescence per cell was determined for 100 cells. PM10 (UK) increases PAFR expression. Data are represented as means and SEMs (n = 50 cells) and are representative of 3 separate experiments. ∗P < .001 by t test. C, Effect of PAFR blocker WEB2086; 10 μmol/mL on PM10 (UK)–stimulated adhesion of S pneumoniae to A549 cells. The amount of adherent bacteria is expressed as CFU. Blocking PAFR attenuates both basal adhesion and the PM10 (UK)–stimulated increase in CFU count. ∗P < .05 versus PM10 without PAFR blocker, by t test. Data are represented as means and SEMs (n = 5) and are representative of >3 separate experiments. Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Lactate dehydrogenase (LDH) release from A549 cells cultured with PM10 (UK). LDH release is indexed to maximum possible LDH release determined after cell lysis (total = 100%). Cells were cultured for 4 hours with either medium alone (control) or with PM10 (UK) at 50 μg/mL. Data are given as means and SEMs (n = 5). LDH release is low from both control and PM-stimulated cells with no significant difference between control and PM10 (UK)–exposed cells (P = NS by t test control vs PM10 treated cells). Journal of Allergy and Clinical Immunology 2011 127, 1236-1242.e2DOI: (10.1016/j.jaci.2010.11.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions