Project title:

Neutrophil polarisation in the airways of infants hospitalised with bronchiolitis

Chief Investigator:

Dr Dani-Louise Dixon

Funding Amount:

$75,000

Recipient:

Flinders University

Overview:

Bronchiolitis is the most common severe respiratory tract illness in infants and remains a major cause of infant hospitalisation. Apart from supportive intervention there is no treatment. Bronchiolitis is predominantly caused by viral infections that induce immune cells (neutrophils) to damage the lung, increasing severity and leading to chronic wheeze in up to 50% of patients. Our lab demonstrated that decreased neutrophil prevalence in the airways is associated with decreased severity of bronchiolitis. However new research suggests that neutrophil type may be as important as absolute number.

Research outcomes:

Researchers:

Dani-Louise Dixon, Mark Lawrence, Suja Mathew

Research Completed:

2017

Research Findings:

Bronchiolitis is the most common severe respiratory tract illness in infants and remains a major cause of infant hospitalisation. Apart from supportive intervention there is no treatment. Bronchiolitis is predominantly caused by viral infections that induce immune cells (neutrophils) to damage the lung, increasing severity and leading to chronic wheeze in up to 50% of patients. Our lab demonstrated that decreased neutrophil prevalence in the airways is associated with decreased severity of bronchiolitis. However, new research suggests that neutrophil type may be as important as absolute number. Our study found that infants with severe bronchiolitis ie requiring supplemental oxygen and/or nasogastric feeds during hospitalization had elevated mediators responsible for neutrophil migration into their airways at admission. However, this was not associated with increased numbers of neutrophils when compared to bronchiolitic infants not requiring interventional therapy. Despite alleviation in clinical symptoms, total airway neutrophils also does not appear to resolve by discharge. However, a significant amount of an immunosuppressive mediator was found which may be responsible for decreasing the inflammatory activity of these neutrophils within the airways of bronchiolitic infants. Further investigation is merited, with analyses over disease course to assess the biological significance.

Key Outcomes:

Aim: Recent studies have identified two distinct subsets of the immune cells, neutrophils, designated: Nl, proinflammatory, and N2, reduced inflammatory. We aimed to profile these neutrophil subsets, as well as specific markers of their recruitment and activation, in nasal washouts (nasopharygeal aspirates; NPA) from infants with bronchiolitis longitudinally across hospital admission to assess the contribution of neutrophil these subsets to disease severity and resolution.

Methods: Daily consecutive NPA samples were collected from hospitalised bronchiolitic infants (:S12 months of age) presenting to Flinders Medical Centre, Adelaide, Australia, between May and September 2015 & 2016. Neutrophil infiltration and activation was assessed via leukocyte differential counts, staining of specific cell surface markers, by immunohistochemistry, and quantification of interleukin (IL)-8, a potent neutrophil chemoattractant, IL-26, a mediator of the IL-8 pathway, myeloperoxidase (MPO), a marker of neutrophil degranulation, and TGF-P 1, an N2 soluble mediator, by ELISA. Augmented clinical severity was attributed to interventional therapy requirement (supplemental oxygen and/or continuous nasogastric feeds) for 2: 24 hours.

Results: NPA samples were obtained from twelve viral-positive bronchiolitic infants. Length of stay (LOS) varied from <1 to 7 days, therefore primary assessment was limited to admission and discharge samples. Interventional therapy (> 24 hours) was required by 7 infants (58%). Age at admission and length of hospitalisation did not differ between severity groups. Median NPA neutrophil percentage of total leukocytes at admission was 88.9%. IL-8 was elevated at admission in infants subsequently requiring interventional therapy (p=0.048). However, IL-26, MPO, total leukocyte and neutrophil concentrations did not differ in NPA admission samples between infants requiring interventional therapy and infants with no interventional therapy requirements. IL-8 concentration at admission did not correlate with either neutrophil concentration at admission or discharge. NPA total leukocyte, neutrophil, IL-8, IL-26 and MPO concentrations did not differ significantly between admission and discharge, irrespective of interventional therapy requirement , nor correlate with LOS.

Concentrations of active TGF-Pl detected were comparable to levels previously identified to significantly suppress neutrophil activity in vitro (> 1 pg/mL). An overall strong positive correlation between TGF- Pl and the percentage of neutrophils stained for N2 marker MMP-9 was observed; with no correlation observed between TGF-Pl and neutrophils positive for Nl marker ICAM-1. Temporal assessment of neutrophil polarization is continuing.

Conclusions: Bronchiolitic infants requiring interventional therapy during hospitalization had elevated markers of airway neutrophil chemotaxis at admission. However, this was not associated with increased neutrophil prevalence or activation when compared to bronchiolitic infants not requiring interventional therapy. Therefore, at admission, IL-8 is apoor predictor of airway neutrophil infiltration across hospitalization. Despite alleviation in clinical symptoms, the total airway neutrophilic response does not appear to resolve by discharge. The detection of pre-established neutrophil polarizing mediators, and the correlation of these mediators with different neutrophilic subsets within NPAs indicates a potential Nl/N2 paradigm occurring within the airways of bronchiolitic infants. Further investigation is merited, with analyses over disease trajectory and by disease severity required to assess biological significance.

Research Papers:

Published:

Cavallaro E, Liang KK, Lawrence MD, Forsyth KD, Dixon DL. The effect of differing viral aetiologies on inflammatory mediators in nasopharyngeal lavage of infants hospitalised with bronchiolitis. Pediatr. Pulmonol. 2016; 52(2):238-246 . doi: 10.1002/ppul.23514.

In Press: (Published Abstracts)

Cavallaro EC, Lawrence MD, Forsyth KD, Dixon DL. A Temporal Analysis of Neutrophil Activity in the Airways of Infants with Bronchiolitis. Am J Resp Crit Care Med, 2017; 195:A (in press)

Cavallaro EC, Liang KK, Forsyth KD, Dixon DL. Neutrophil polarization in the airways  of infants with bronchiolitis.  JImmunol  (in press)

In Preparation:

Cavallaro EC, Liang KK, Forsyth KD, Dixon DL. IL-26 in the airways of children hospitalised with bronchiolitis.

Cavallaro EC, Lawrence MA, Forsyth KD, Dixon DL. Neutrophil Polarisation in the Airways of Infants with Bronchiolitis.

Conference presentations arising from this research project.

Cavallaro EC, Lawrence MA, Liang KK, Mathew SM, Forsyth KD, Dixon DL.  Neutrophil Polarisation in the Airways of Infants with Bronchiolitis.  The Australian Society for Medical Research:  South Australian Division. Adelaide, 2016

Liang KK, Cavallaro E, Dixon DL. IL-26 in the airways of children hospitalised with bronchiolitis: a novel discovery. Australian Society for Medical Research: South Australian Division.  Adelaide, 2016

Cavallaro EC, Lawrence MD, Forsyth KD, Dixon DL.  A Temporal Analysis of Neutrophil Activity in the Airways oflnfants with Bronchiolitis.   American Thoracic Society, Washington DC, USA, 2017.

Cavallaro EC, Liang KK, Forsyth KD, Dixon DL. Neutrophil polarization in the airways of infants with bronchiolitis .  The American Association of Immunologists, Washington DC, USA, 2017.

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