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Ciliated Cultures From Patients With Primary Ciliary Dyskinesia Produce Nitric Oxide in Response to Haemophilus influenzae Infection and Proinflammatory CytokinesPrimary Ciliary Dyskinesia Epithelia Nitric Oxide FREE TO VIEW

Woolf T. Walker, BM; Claire L. Jackson, PhD; Janice Coles, BSc (Hons); Peter M. Lackie, PhD; Saul N. Faust, MBBS, PhD; Luanne Hall-Stoodley, PhD; Jane S. Lucas, BM, PhD
Author and Funding Information

From the Primary Ciliary Dyskinesia Centre (Drs Walker, Jackson, Coles, Lackie, and Lucas), and Southampton NIHR Respiratory Biomedical Research Unit and NIHR Wellcome Trust Clinical Research Facility (Drs Walker, Faust, Hall-Stoodley, and Lucas), University Hospital Southampton NHS Foundation Trust; Academic Unit of Clinical and Experimental Sciences (Drs Walker, Jackson, Coles, Lackie, Faust, Hall-Stoodley, and Lucas), Faculty of Medicine, University of Southampton; and the Department of Microbial Infection and Immunity (Dr Hall-Stoodley), The Ohio State University, Columbus, OH.

Correspondence to: Jane S. Lucas, BM, PhD, Academic Unit of Clinical and Experimental Sciences (Mail Point 803), University of Southampton Faculty of Medicine, University Hospital Southampton NHS Foundation Trust, Tremona Rd, Southampton, SO16 6YD, England; e-mail: jlucas1@soton.ac.uk


Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2014;145(3):668-669. doi:10.1378/chest.13-2398
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To the Editor:

We read with interest the article by Smith et al1 in an issue of CHEST (November 2013) examining nitric oxide (NO) biosynthesis in primary ciliated epithelial cell cultures from patients with primary ciliary dyskinesia (PCD) at baseline and 2 h after coculture with Pneumococcus, reporting no increase in NO. We have investigated the modulation of NO in primary cultured ciliated epithelial cells from patients with PCD following 72-h coculture with nontypeable Haemophilus influenzae (NTHi) isolated from infected patients with PCD and following stimulation with proinflammatory cytokines. Our study was designed to investigate NTHi biofilm development on cells from patients with PCD as opposed to the investigation of acute pneumococcal infection in Smith et al’s1 article. The study was approved by the Southampton and South West Hampshire Research Ethics Committee (06/Q1702/109).

Airway epithelium from patients with PCD and control patients without PCD was obtained by nasal brushing and was cultured at the air-liquid interface until differentiated and ciliated. We quantified the presence of NO using a total NO detection assay (Enzo Life Sciences, Inc) within phosphate-buffered saline washes applied to the apical surface of air-liquid interface cultures. NO levels were measured before and after epithelial cells were apically cocultured for 72 h with NTHi at a multiplicity of infection of 100 to evaluate biofilm infection. Cell viability was demonstrated by daily stable transepithelial electrical resistance (PCD and non-PCD) and ciliary beat frequency measurements (control subjects without PCD). We also measured levels following an 18-h incubation with a cocktail of proinflammatory cytokines (10 ng/mL each of IL-1β/interferon-γ/tumor necrosis factor-α) applied basolaterally to the cells (n = 14 for each experiment).

Our baseline data were consistent with that of Smith et al,1 demonstrating no difference between the NO levels from PCD and non-PCD epithelia (Fig 1, Table 1). However, we found a significant twofold to threefold increase in NO levels from both PCD and non-PCD epithelia in response to NTHi and proinflammatory cytokines, with no significant difference between the two patient groups (Fig 1, Table 1, e-Fig 1). We corroborated basal NO biosynthesis in PCD and non-PCD epithelial cell cultures using 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate fluorescence (data not shown).

Figure Jump LinkFigure 1. Nitric oxide levels measured in apical phosphate-buffered saline washes following a 30-min incubation at 37°C of ciliated primary epithelial cell layers from primary ciliary dyskinesia (PCD) (n = 5) and non-PCD (n = 9) cultured at an air-liquid interface, measured at baseline and 72-h post coculture with nontypeable Haemophilus influenzae (mean ± SEM). *P < .05. ● = PCD; ▪ = non-PCD. NS = nonsignificant.Grahic Jump Location
Table Graphic Jump Location
Table 1 —NO Levels Biosynthesized From PCD vs Non-PCD Ciliated Epithelia

NO levels biosynthesized from PCD vs non-PCD ciliated epithelia cultured at an air-liquid interface at baseline and following stimulation with either 10 mg/mL IL-1β/IFN-γ/TNF-α for 18 h or coculture with NTHi for 72 h (mean ± SEM). IFN = interferon; NO = nitric oxide; NTHi = nontypeable Haemophilus influenzae; PCD = primary ciliary dyskinesia; TNF = tumor necrosis factor.

We speculate that the longer period of infection (72 h compared with 2 h) might account for the differences between our data and that of Smith et al.1 The difference in organism (NTHi from infected patients with PCD rather than a laboratory strain of Pneumococcus) might also contribute to the difference. Taking these data alongside the findings of Smith et al,1 we suggest that NO biosynthesis in nasal epithelia from patients with PCD may be delayed in response to infection. Results of these two studies highlight the need to investigate NO response of PCD epithelial cells to different pathogens over a range of time points. The etiology for the significantly reduced nasal NO levels in patients with PCD remains unanswered.2

Acknowledgments

Additional information: The e-Figure can be found in the “Supplemental Materials” area of the online article.

Smith CM, Fadaee-Shohada MJ, Sawhney R, et al. Ciliated cultures from patients with primary ciliary dyskinesia do not produce nitric oxide or inducible nitric oxide synthase during early infection. Chest. 2013;144(5):1671-1676. [CrossRef] [PubMed]
 
Walker WT, Jackson CL, Lackie PM, Hogg C, Lucas JS. Nitric oxide in primary ciliary dyskinesia. Eur Respir J. 2012;40(4):1024-1032. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1. Nitric oxide levels measured in apical phosphate-buffered saline washes following a 30-min incubation at 37°C of ciliated primary epithelial cell layers from primary ciliary dyskinesia (PCD) (n = 5) and non-PCD (n = 9) cultured at an air-liquid interface, measured at baseline and 72-h post coculture with nontypeable Haemophilus influenzae (mean ± SEM). *P < .05. ● = PCD; ▪ = non-PCD. NS = nonsignificant.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 —NO Levels Biosynthesized From PCD vs Non-PCD Ciliated Epithelia

NO levels biosynthesized from PCD vs non-PCD ciliated epithelia cultured at an air-liquid interface at baseline and following stimulation with either 10 mg/mL IL-1β/IFN-γ/TNF-α for 18 h or coculture with NTHi for 72 h (mean ± SEM). IFN = interferon; NO = nitric oxide; NTHi = nontypeable Haemophilus influenzae; PCD = primary ciliary dyskinesia; TNF = tumor necrosis factor.

References

Smith CM, Fadaee-Shohada MJ, Sawhney R, et al. Ciliated cultures from patients with primary ciliary dyskinesia do not produce nitric oxide or inducible nitric oxide synthase during early infection. Chest. 2013;144(5):1671-1676. [CrossRef] [PubMed]
 
Walker WT, Jackson CL, Lackie PM, Hogg C, Lucas JS. Nitric oxide in primary ciliary dyskinesia. Eur Respir J. 2012;40(4):1024-1032. [CrossRef] [PubMed]
 
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