Anti-inflammatory Deficiencies in Neutrophilic Asthma

Reduced Galectin-3 and IL-1RA/IL-1 Beta

Peng Gao; Peter G Gibson; Katherine J Baines; Ian A Yang; John W Upham; Paul N Reynolds; Sandra Hodge; Alan L James; Christine Jenkins; Matthew J Peters; Jie Zhang; Jodie L Simpson


Respiratory Research. 2015;16(5) 

In This Article


Gal-3 and gal-3BP in Asthma Inflammatory Phenotypes

Participants' sex, age, BMI, smoking history, atopy and ACQ6 were similar among the four asthma subgroups [Table 1]. Participants with PGA had the best lung function compared to other groups. Those with NA had the greatest sputum total cell count and number of neutrophils compared with the other groups, followed by MGA. There were higher numbers of eosinophils in MGA and EA than in NA and PGA, while EA had a decreased number of macrophages and lymphocytes compared with PGA and MGA respectively.

Serum gal-3 and gal-3BP were similar across the inflammatory phenotypes [Table 1]. Due to the small numbers of participants with MGA further analyses are conducted among those with NA, PGA and EA.

Inflammatory Mediators by Asthma Inflammatory Subtype

Sputum gal-3 was significantly reduced in NA compared with PGA and EA and gal-3BP was increased in NA compared with EA. The gal-3 to gal-3BP ratio was significantly reduced in participants with NA compared with EA and PGA. Participants with NA also had significantly increased concentrations of sputum IL-1β, IL-6 and IL-8 compared with PGA and EA. Although the level of IL-1RA was not different between the asthma inflammatory subtypes, the ratio of IL-1RA to IL-1β was significantly lower in NA compared with EA and PGA [Figure 1].

Figure 1.

Induced sputum concentrations of inflammatory mediators in asthmatic inflammatory subtypes. Gal-3 (A) was decreased, gal-3BP (B) was increased and the gal-3 to gal-3BP ratio (C) was decreased in NA. IL-1RA (D) was not different, IL-1β (E) was increased and the IL-1RA to IL-1β ratio (F) was reduced in NA. IL-6 (G) and IL-8 (H) were both increased in NA. Group comparisons were conducted using analysis of variance with least significant difference (LSD) post hoc test after being log-transformed and adjusted by age and BMI. The horizontal bar denotes median value. NA; neutrophilic asthma, PGA; paucigranulocytic asthma, EA; eosinophilic asthma.

Association Between Inflammatory Mediators and Clinical Characteristics

Sputum gal-3 was negatively associated with the total number of sputum cells, the number of sputum neutrophils and lymphocytes while gal-3BP was positively associated with sputum total cells, neutrophils, macrophages and lymphocytes [Table 2].

Sputum IL-1β, IL-8 and IL-6 were all positively associated with the sputum total cell count, number of neutrophils and lymphocytes. While sputum IL-6 was significantly associated with the number of sputum macrophages. There were no significant relationships between IL-1RA and sputum inflammatory cell counts (data not shown). None of the sputum markers were associated with sputum eosinophil number (all p > 0.05).

Gal-3 had a significant inverse association with IL-1β and positive association with IL-6. Gal-3BP was significantly positively associated with IL-1β, IL-6, and IL-8. The typical pro-inflammatory mediators such as IL-1β, IL-6, and IL-8 were positively associated with each other. There were no significant correlations between IL-1RA and other mediators (data not shown).

Only IL-1RA had a significant relationship with FEV1 (r = 0.386, p < 0.001) and FVC (r = 0.332, p < 0.001) Table 3.

In participants with neutrophilic asthma sputum gal-3 levels were significantly positively associated with mean ACQ score (spearman's r = 0.500, p = 0.049) but not with FEV1% predicted or FEV1/FVC (data not shown). There were no significant associations between sputum gal-3BP or the gal-3/gal-3BP with ACQ, FEV1 % predicted for FEV1/FVC (data not shown).

Gal-3 and gal-3BP in Sputum Cells

Gal-3 and gal-3BP immunoreactivity was observed in sputum macrophages from all subtypes of asthma [Figure 2i]. Macrophages in neutrophilic asthma appeared to have less gal-3 staining compared with both eosinophilic and paucigranulocytic asthma and when gal-3 was present, it was colocalized with gal-3BP. Macrophages in EA and PGA appeared to have very intense staining for gal-3 and markedly less gal-3BP immunoreactivity. In the sputum neutrophils, there appeared to be less nuclear gal-3 in the participant with NA compared with EA and PGA [Figure 2ii].

Figure 2.

Sputum cells from; A) neutrophilic, B) eosinophilic and C) paucigranulocytic asthma stained for gal-3 (green) and gal-3BP (red) with nuclear DAPI.

Logistic Regression Analysis to Identify Predictors of Asthma Phenotypes

The ratio of gal-3 to gal-3BP was an independent predictor of the presence of NA from EA and PGA after correcting for age, sex and BMI. The model reached statistical significance with an adjusted R2 of 0.322, p = 0.019. Full details of the regression can be found in Table S1 of Additional file 2

Effect of Asthma Medication

We compared the sputum inflammatory mediator levels in those taking ICS only with those taking combination ICS/LABA therapy. Sputum IL-β levels were very low in those participants taking ICS only and significantly reduced compared to ICS/LABA. There were no differences in any other mediators. Similarly, there was no difference in any mediator in those participants taking very high doses of ICS compared with those taking lower doses of ICS [Table 4].

We then conducted a multiple linear regression to investigate the factors that are independently associated with sputum gal-3 levels. The dose of ICS was not associated with sputum gal-3 levels (p = 0.289) and therefore was not included in the final model. The model was highly significant with BMI, sputum bronchial epithelial cell proportion, sputum IL-1β and previous smoking being independently associated with sputum gal-3 levels [Table 5].