Impact of Sex on Circulating Leukocytes Composition in COPD Patients

Abstract

Purpose: Chronic obstructive pulmonary disease is characterized by chronic inflammatory response both at the lung site and at the systemic level. Abnormalities in circulating leukocytes have been reported to occur in COPD patients and have been often shown to correlate with the decline in lung function. COPD affects men and women at a virtually comparable rate, even though distinct sex specific symptoms, progression and therapeutic implications have been described. Nonetheless, these sex-associated differences have not been analyzed in terms of circulating leukocytes. To assess the impact of sex on the changes of circulating immune cells in COPD patients.

Patients and methods: Blood samples were collected from 50 COPD patients (31 males, 19 females) and 63 age and sex-matched controls (35 males, 28 females) enrolled in this pilot study. Complete blood cell count and multi-parametric flow cytometry analysis were performed to characterize the leukocyte populations and subsets.

Results: Male COPD patients are distinguished from controls by a significant increase in white blood cell counts, neutrophil total and differential counts, and neutrophil-to-lymphocyte ratio. Conversely, a generalized leukocyte decrease discriminated female COPD patients from the related controls. The impact of sex is further remarked by a decrease in adaptive immune cell subpopulations in males as opposed to a consistent increase of innate immune cell types in females correlating with disease severity.

Conclusion: These data indicate that the definition of specific changes of circulating leukocytes to be used as reliable biomarkers of the disease severity cannot be accomplished irrespectively of sex.

Keywords: COPD; blood leukocytes; neutrophil-to-lymphocyte ratio; neutrophilia; sex.

Figure 1

Sex-specific alteration of circulating leukocytes in COPD patients and controls. White blood cells (WBC), neutrophils (N), lymphocytes (L), monocytes (M), eosinophils (E), basophils (B) total counts in controls (ctrl, empty dots) and COPD patients (black dots) complete cohorts (A), male cohort (C) and female cohort (E). Differential counts of neutrophils (N), lymphocytes (L), monocytes (M), eosinophils (E), basophils (B) in controls (empty dots) and COPD patients (black dots) complete cohorts (B), male cohort (D) and female cohort (F). The red line represents the median. *P < 0.05, **P < 0.01 according to Mann Whitney or Student’s t-test.

Figure 2

Characterization of monocyte and dendritic cell subtypes populations in COPD patients and controls. Frequency of classical monocytes, monocytic-myeloid derived suppressor cells (M-MDSC), intermediate monocytes and non-classical monocytes in total monocytes are shown for male (A) and female (B) COPD patients and controls (ctrl). In addition, total DCs (C and E), as well as the frequency of plasmacytoid (pDC), conventional DC1 (cDC1) and DC2 (cDC2) in DCs (D and F) are shown for male (C and D) and female (E and F) COPD and control donors. Controls: empty dots, COPD: black dots. The red line represents the median.

Figure 3

Characterization of lymphoid populations in COPD patients and controls. (A and B) T lymphocytes, B lymphocytes, NK cells and NKT cells counts in male (A) and female (B) COPD and control (ctrl) donors. (C and D) Differential counts of T helper cells (CD3⁺CD4⁺, Th) and cytotoxic T-lymphocytes (CD3⁺CD8⁺, CTLs) in total T cells in male (C) and female (D) COPD and control donors. (E and F) Differential counts of Th1, Th2, Th17, Th1/Th17 and regulatory T cells (Treg) in total T helper cells in male (E) and female (F) COPD and control donors. Controls: empty dots, COPD: black dots. The red line represents the median. *P < 0.05 according to Mann Whitney test, **P < 0.01 according to Student’s t-test.

Figure 4

Impact of smoking and comorbidities on leukocyte parameters in male and female COPD patients. Heat map representation of the ANOVA analysis in male (A) and female (B) COPD patients and sex matched controls. P-value obtained from the ANOVA analysis for COPD, smoking, CAD, hypertension or arthrosis alone or interacting with COPD (interaction) are plotted according to the scale shown on the right. The smoking impact was evaluated by grouping donors according to smoking index (pack/year) in: “very light” (pack/year < 15); “light” (pack/year 15–30), and “heavy” (pack/year ≥ 30).

Figure 5

Correlation of leukocyte populations with parameters of COPD severity. Correlation analysis between all leukocyte populations and FEV_1% of predicted, FEV₁/FVC or GOLD was performed separately for male and female COPD patients. Heatmap shows all leukocyte populations correlating in a statistically significant manner (P < 0.05) with at least one clinical parameter in male (left) or female (right) COPD patients. The color scale indicates the degree of the correlation: blue = leukocyte amount decreases with COPD severity; red = leukocyte amount increases with diseases severity. Raw Spearman or Pearson correlation coefficient is reported inside the heatmap.

Figure 6

Correlation of leukocyte populations with COPD severity parameters in male COPD patients. Correlation between: WBC (A) or lymphocyte count (B) with FEV₁/FVC; B cell count with FEV₁% (C), FEV1/FVC (D) and GOLD (E); NKT cell count with FEV₁% (F), FEV₁/FVC (G) and GOLD (H); cDC2 frequency with GOLD (I). Linear regression and 95% confidence interval are drawn. Spearman of Pearson correlation coefficient (r) and p-value (P) are shown.

Figure 7

Correlation of leukocyte populations with COPD severity parameters in female COPD patients. Correlation between: Th1 cell frequency with FEV_1% (A) and GOLD (B); Treg cell frequency with FEV₁% (C) and GOLD (D); eosinophil count with FEV_1% of predicted (E) and GOLD (F); basophil count with GOLD (G); cDC1 frequency with FEV1% (H), GOLD (I) and FEV₁/FVC (J); M-MDSC frequency with FEV₁% (K), GOLD (L), and FEV₁/FVC (M); pDC count with FEV₁% (N). Linear regression and its 95% confidence interval are drawn. Spearman or Pearson correlation coefficient (r) and p-value (P) are shown.

Figure 8

Dynamic changes in circulating leukocytes in different stages of COPD in males and females. Graphs represent the ratio COPD/controls for granulocytes and lymphocytes. In male COPD (A), a significant increase in neutrophils compared to controls, is detectable at early stages and it is maintained elevated throughout all the disease stages, while a decrease in lymphocyte number can be observed only in more severe male patients. In females (B), hallmark of COPD is represented by a significant reduction in lymphocyte number compared to controls, and an increase in eosinophils and basophils can be observed only as the disease progresses.