Measures of gene expression in sputum cells can identify TH2-high and TH2-low subtypes of asthma

Abstract

Background: The 3-gene signature of periostin, chloride channel accessory 1 (CLCA1), and Serpin β2 (SERPINB2) in airway epithelial brushings is used to classify asthma into TH2-high and TH2-low endotypes. Little is known about the utility of gene profiling in sputum as a molecular phenotyping method.

Objective: We sought to determine whether gene profiling in sputum cells can identify T(H)2-high and T(H)2-low subtypes of asthma.

Methods: In induced sputum cell pellets from 37 asthmatic patients and 15 healthy control subjects, PCR was used to profile gene expression of the epithelial cell signature of IL-13 activation (periostin, CLCA1, and SERPINB2), TH2 genes (IL4, IL5, and IL13), and other genes associated with airway TH2 inflammation.

Results: Gene expression levels of CLCA1 and periostin, but not SerpinB2, were significantly higher than normal in sputum cells from asthmatic subjects. Expression levels of IL-4, IL-5, and IL-13 were also significantly increased in asthmatic patients and highly correlated within individual subjects. By combining the expression levels of IL-4, IL-5, and IL-13 in a single quantitative metric ("T(H)2 gene mean"), 26 (70%) of the 37 asthmatic patients had T(H)2-high asthma, which was characterized by more severe measures of asthma and increased blood and sputum eosinophilia. TH2 gene mean values tended to be stable when initial values were very high or very low but fluctuated above or below the T(H)2-high cutoff when initial values were intermediate.

Conclusion: IL-4, IL-5, and IL-13 transcripts are easily detected in sputum cells from asthmatic patients, and their expression levels can be used to classify asthma into T(H)2-high and T(H)2-low endotypes.

Keywords: Asthma; BME; CLCA1; CPA3; Carboxypeptidase A3; Chloride channel accessory 1; Feno; Fraction of exhaled nitric oxide; IL-13; IL-17; IL-4; IL-5; LABA; Long-acting β-agonist; RIN; RNA integrity number; Serpin β2; SerpinB2; T(H)2 cell; UCSF; University of California, San Francisco; cytokines; eosinophils; eotaxin; inflammation; mast cells; phenotypes; sputum; β-Mercaptoethanol.

FIG 1

A, RIN values are highest in sputum cells stored and processed with optimized methods. *Significant difference between methods (P < .01). B, Expression of periostin and CLCA1 is higher than normal in asthmatic patients, but expression of SerpinB2 is not. C, IL4, IL5, and IL13 expression is higher than normal in asthmatic patients, but expression of IL17 and IFNG is not. *Significant difference from healthy control subjects (P < .0001).

FIG 2

The T_H2 gene mean as a scaled and centered mean expression value for IL-4, IL-5, and IL-13. The T_H2 gene mean data for the healthy subjects show the cutoff value that represents 2 SDs greater than the mean value. ICS, Inhaled corticosteroids. *Significant difference between asthmatics on ICS and not on ICS compared to healthy controls (P < .01).

FIG 3

A, The T_H2 gene mean is highly reproducible in RNA aliquots of induced sputum from asthmatic patients. B, The T_H2 gene mean in 13 asthmatic patients at repeat visits. Red lines, Persistent T_H2-high asthma; black lines, intermittent T_H2-high asthma; blue lines, persistent T_H2-low asthma. Triangles represent subjects taking inhaled corticosteroids, and circles represent subjects not taking inhaled corticosteroids.

FIG 4

The T_H2 gene mean is significantly higher in patients with poorly controlled asthma (Asthma Control Test [ACT] score <20 or prebronchodilator FEV₁ percent predicted <80%), whereas IL17 gene expression is not. The T_H2 gene mean is inversely correlated with prebronchodilator FEV₁ percent predicted, but it is not correlated with IL17 gene expression. *Significant difference from healthy control subjects (P < .01).

FIG 5

Gene expression for eotaxin genes (CCL11, CCL24, and CCL26) and mast cell genes (tryptase, CPA3, and chymase) in sputum cells from patients with T_H2-high and T_H2-low asthma. *Significant difference from healthy control subjects (P < .01).

FIG 6

Receiver operating characteristic (ROC) analysis of peripheral blood eosinophil counts, sputum eosinophil counts, and Feno levels as biomarkers of airway T_H2 inflammation, as assessed by using the sputum cell T_H2 gene mean. Green line, ROC curve for peripheral blood eosinophil counts; blue line, ROC for sputum eosinophil counts; red line, ROC for Feno values. AUC, Area under the curve.

FIG E1

Gene expression for IL4, IL5, and IL13 in sputum cells is highly correlated among asthmatic patients. Data are log₂-normalized gene expression.

FIG E2

Influence of the time interval between sputum induction on the reproducibility of T_H2 classification. Red lines, Persistent T_H2-high asthma; black lines, intermittent T_H2 asthma; blue lines, persistent T_H2-low asthma. Triangles represent subjects taking inhaled corticosteroids, and circles represent subjects not taking inhaled corticosteroids.