Abnormal M1/M2 macrophage phenotype profiles in the small airway wall and lumen in smokers and chronic obstructive pulmonary disease (COPD)

Abstract

We explore potential dysregulation of macrophage phenotypes in COPD pathogenesis through integrated study of human small airway tissue, bronchoalveolar lavage (BAL) and an experimental murine model of COPD. We evaluated human airway tissue and BAL from healthy controls, normal lung function smokers (NLFS), and COPD subjects. Both small airways and BAL cells were immunohistochemically stained with anti-CD68 for total macrophages and with anti-CD163 for M2, and anti-iNOS for M1 macrophages. Multiplex ELISA measured BAL cytokines. Comparable cigarette smoke-induced experimental COPD mouse model was assessed for relevant mRNA profiles. We found an increase in pro-inflammatory M1s in the small airways of NLFS and COPD compared to controls with a reciprocal decrease in M2 macrophages, which remained unchanged among pathological groups. However, luminal macrophages showed a dominant M2 phenotype in both NLFS and COPD subjects. BAL cytokine skewed towards an M2 profile with increase in CCL22, IL-4, IL-13, and IL-10 in both NLFS and COPDs. The mouse-model of COPD showed similar increase in mRNA for M2 markers. Our finding suggests abnormal macrophage switching in both mucosal and luminal areas of COPD patients, that strongly associated with cytokine balance. There may be potential for beneficial therapeutic cytokine manipulation of macrophage phenotypes in COPD.

Figure 1

Macrophages phenotype numbers in SA tissue. In epithelium: an increase in the numbers of (a) M1 and a decrease in (b) M2 macrophages observed in smokers and COPDs compared to NC. Similar pattern was also observed in the sub-epithelium (d) M1 and (e) M2. Percent change in macrophage phenotypes M0, M1 and M2 in (c) Epithelium and (f) Sub-epithelium expression (Data in median and range; *p < 0.05, **p < 0.01).

Figure 2

Representative micrographs of M1 macrophages dual stained for iNOS (brown) and CD68 (blue). (a) Thin epithelium and thin walled normal control, (b) thick epithelium and thick walled COPD-CS, counterstained with nuclear fast red (pink). () Dual stained CD68 + iNOS + cells, () only CD68 + cells. CD163 staining M2 macrophages (c) NC (d) COPD-CS.

Figure 3

Regression analysis for tissue macrophage phenotypes with pack year history for NLFS and COPD-CS for (a) epithelial M1 and (b) sub-epithelial M2 macrophages.

Figure 4

Arginase-1 expression in the small airway wall of (a) NC compared to (b) COPD-CS. A significant increase in Arginase-1 expression was observed in both (c) epithelium and (d) sub-epithelium. Data are presented as median and range; group comparisons with Mann-Whitney two-tailed t-test; p < 0.05 was considered significant.

Figure 5

Expression patterns of alveolar macrophages (AMs) in the alveolar spaces in resected tissue of COPD patients (200x), (a) M1 AMs dual stained for iNOS (brown) and CD68 (blue), counterstained with nuclear fast red (pink), M2 phenotype macrophages stained brown with (b) CD163 and (c) arginase-1(ARG-1), counterstained with nuclear-stained hematoxylin (blue).

Figure 6

Representative pictures (400x) of M1 AMs dual stained for iNOS (brown) and CD68 (blue); (a) Normal control, and (b) COPD-CS, counterstained with nuclear fast red (pink). M2 phenotype macrophages stained brown with CD163 and arginase-1(ARG-1), (c,e) Normal controls and (d,f) COPD-CS respectively, with nuclear-stained hematoxylin (blue).

Figure 7

AMs numbers in BAL. (a) Total AMs, (b) M1 and (c) M2 macrophages in NC, NLFS COPD-CS, and ES. (d) Represent percent of total macrophage for each phenotypic population (Data in median and range; ***p < 0.001, *p < 0.05).

Figure 8

Regression analysis for BAL AMs with lung function in COPD groups. (a) Total AMs and (b) M2 AMs.

Figure 9

Cytokine profiles in BAL of NC, NLFS, COPD-CS and ES: (a) M1, (b) M2, (c) inflammatory cytokines (IL-6, IL-1β and TNFα) (d) IL-12/IL-4 ratio.

Figure 10

Positive correlation of M2 macrophages with (a) CCL22 and (b) IL-4 in COPD-CS.

Figure 11

Fold changes in mRNA levels of M1 and M2 chemokines in the lungs of mice chronically exposed to cigarette smoke for (a) six and (b) eight weeks and normal air exposed controls (**p < 0.0.01; *p < 0.05).