Inflammatory leukocyte phenotypes correlate with disease progression in idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive deposition of extracellular matrix, worsening dyspnea, and eventual mortality. Pathogenesis of IPF is poorly understood and the role inflammation and activated leukocytes play in the disease process is controversial. Previous studies demonstrated that activated leukocyte subsets characterize IPF patients. We sought to validate this observation in a well-defined cohort of 35 IPF patients and to correlate the observed leukocyte phenotypes with robust parameters of disease progression. We demonstrate that in univariate and multivariate analyses, increases in the CD14hi, CD16hi subset of monocytes measured at baseline correlated with disease progression, with a threshold value >0.5% of the total peripheral blood mononuclear cells being a significant predictor for worse outcome. In addition, several T cell subsets, including CD25 expressing CD4 cells, and CXCR3 expressing CD4 and CD8 subsets correlated with disease progression when found in increased percentages in the peripheral blood of IPF patients when sampled at baseline. Somewhat surprising in comparison to previous literature, the CD4 T cells did not appear to have lost expression of the co-stimulatory molecule, CD28, but the CD8 T cells did. Taken together, these results are consistent with the presence of an inflammatory process in IPF patients who eventually progress. However, when longitudinal measurements of these same markers were examined, there was significant heterogeneity of expression and these biomarkers did not necessarily remain elevated in IPF patients with progressive disease. We interpret this heterogeneity to suggest that IPF patients experience episodic inflammatory events that once triggered, may lead to disease progression. This longitudinal heterogeneity in biomarker analyses may explain why such markers are not consistently measured in all IPF cohorts.

Keywords: interstitial lung disease; lung; lymphocyte; monocyte; peripheral blood.

Figure 1

Progressive IPF patients are characterized by higher percentages of CD4 CD25+ and CD8 CXCR3+ cells. The box shows the range of the data from 25th percentile to 75th percentile. The bar in the middle of the box represents the median (50th percentile). The lower and upper whiskers gives the range of the data with each whisker restricted to 1.5 times the interquartile range (which is defined as the upper quartile minus the lower quartile) with circles indicating data outside of this whisker range. (A) CD4 CD25 percentages are graphed; (B) CXCR3 CD8 percentages are graphed.

Figure 2

Idiopathic pulmonary fibrosis patients have higher percentages of CXCR3 than CCR4 expressing CD4 T cells. Mean values of each cell type were analyzed by unpaired T test adjusting for multiple comparisons using the Holm–Sidak method. (A) graphs CD4 cells while (B) graphs CD8 cells.

Figure 3

Kaplan–Meier plots show decreased probability of progression-free (PF) survival for patients with elevated percentages of T cell and monocyte subsets above identified thresholds. Patients with elevated percentages of (A) CD14hi CD16lo CCR2+ monocytes, (B) CD14hi, CD16hi, CD64+ monocytes, (C) CD4 CD25 T cells, and (D) CXCR3 CD8 T cells above the indicated thresholds identified in Table 3 show worse probability of PF survival.

Figure 4

Longitudinal analyses of T cell phenotypes are highly variable. The percentage of CD4 CD25 (A) or CD8 CXCR3 (B) expressing T cells identified at longitudinal time points (weeks 0, 16, 32, 46, 64, and 80) were graphed. The dotted red line indicates the threshold above which this cell population was found to predict the patient reaching the combined endpoint in Table 5. For both of these markers, univariate and multivariate analyses of the percentages at baseline significantly separated non-progressors from progressors. However, elevation of the marker at baseline, did not predict stable elevation of the marker over time.

Figure 5

Longitudinal analyses of monocyte phenotypes are highly variable. The percentage of CD14hi CD16hi CD64 (top) or CD14hi CD16lo CD64 (bottom) expressing monocytes identified at longitudinal time points (weeks 0, 16, 32, 46, 64, and 80) were graphed. The dotted red line indicates the threshold above which this cell population was found to predict the patient reaching the combined endpoint in Table 5.