Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease - PubMed (original) (raw)

Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease

Renat Shaykhiev et al. J Immunol. 2009.

Abstract

When exposed to a specific microenvironment, macrophages acquire either M1- or M2-polarized phenotypes associated with inflammation and tissue remodeling, respectively. Alveolar macrophages (AM) directly interact with environmental stimuli such as cigarette smoke, the major risk factor for chronic obstructive pulmonary disease (COPD), a disease characterized by lung inflammation and remodeling. Transcriptional profiling of AM obtained by bronchoalveolar lavage of 24 healthy nonsmokers, 34 healthy smokers, and 12 COPD smokers was performed to test the hypothesis whether smoking alters AM polarization, resulting in a disease-relevant activation phenotype. The analysis revealed that AM of healthy smokers exhibited a unique polarization pattern characterized by substantial suppression of M1-related inflammatory/immune genes and induction of genes associated with various M2-polarization programs relevant to tissue remodeling and immunoregulation. Such reciprocal changes progressed with the development of COPD, with M1-related gene expression being most dramatically down-regulated (p < 0.0001 vs healthy nonsmokers, p < 0.002 vs healthy smokers). Results were confirmed with TaqMan real-time PCR and flow cytometry. Among progressively down-regulated M1-related genes were those encoding type I chemokines CXCL9, CXCL10, CXCL11, and CCL5. Progressive activation of M2-related program was characterized by induction of tissue remodeling and immunoregulatory genes such as matrix metalloproteinase (MMP)2, MMP7, and adenosine A3 receptor (ADORA3). Principal component analysis revealed that differential expression of polarization-related genes has substantial contribution to global AM phenotypes associated with smoking and COPD. In summary, the data provide transcriptome-based evidence that AM likely contribute to COPD pathogenesis in a noninflammatory manner due to their smoking-induced reprogramming toward M1-deactivated, partially M2-polarized macrophages.

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Figures

Figure 1

Figure 1

Smoking-mediated reciprocal induction of M1 and M2 polarization programs of human alveolar macrophages. A. Volcano plot of M1-related probe sets significantly differently expressed between healthy nonsmokers (n=24) and healthy smokers (n=34). B. Volcano plot for the M2-related gene probe sets comparing the same groups. For both panels, the x-axis corresponds to the fold-change and the y-axis corresponds to p value. Red dots represent significant differentially expressed probe sets, grey dots represent probe sets with no significant difference between healthy smokers and healthy nonsmokers. The changes in gene expression were considered statistically significant based on the criteria of fold-change ≥1.5, p<0.05 with Benjamini-Hochberg correction.

Figure 2

Figure 2

Biologic phenotypes of healthy smokers compared to healthy nonsmokers based on alveolar macrophage M1- and M2-related gene expression. A. Expression of all M1-related probe sets in AM of healthy nonsmokers (n=24; left panels) compared to that of healthy smokers (n=34; right panels). See Table II for a list of all M1-related probes. B. Expression of all M2-related probe sets in AM comparing the same groups (healthy nonsmokers, left; healthy smokers, right). See Table III for a list of all M2-related probes. For both A and B, the y-axis indicates normalized relative expression levels for the probe sets, the x-axis shows the individuals belonging to each group randomly ordered but of similar order in A and B. Red = gene probe sets down-regulated in healthy smokers as compared to healthy nonsmokers; blue = gene probe sets up-regulated in healthy smokers as compared to healthy smokers. Intensity of color indicates the degree of down- or up-regulation. Note that overall, the M1-related genes tend to be down-regulated in the healthy smokers compared to the healthy nonsmokers. The opposite is observed among the M2-related genes, but not to the same extent as the down-regulation of the M1 genes. C. Non-supervised hierarchical cluster analysis of AM M1- and M2-related gene expression of healthy nonsmokers and healthy smokers. The analysis is based on healthy smokers, the differential expression of M1- and M2- related genes of the same groups of healthy nonsmokers (n=24) and healthy smokers (n=34) using Spearman correlation as a similarity measure and an average linkage as a clustering algorithm. Statistically significant differentially expressed M1- and M2-gene probe sets were used as input data set. Genes expressed above average are represented in red, below average in blue, and average in white. The genes are represented vertically, and individual subjects horizontally at the bottom. Healthy nonsmokers are indicated by green, healthy smokers by orange. Although, there is variability within each group, compared to the healthy nonsmokers, the general tendency for the healthy smokers is for the M1 genes to be down-regulated and the M2 genes up-regulated.

Figure 3

Figure 3

Progressive reciprocal alteration of M1- and M2-related gene expression in alveolar macrophages with the development of COPD. A. Normalized expression levels of all M1-related probe sets; B. M1-related probe sets significantly differentially expressed in AM of COPD smokers vs healthy nonsmokers; C. All M2-related probe sets; and D. M2-related probe sets significantly differentially expressed in AM of COPD smokers vs healthy nonsmokers. The data is based on healthy nonsmokers (n=24), healthy smokers (n=34) and COPD smokers (n=12). The y-axis indicates mean normalized, expression levels for the probe sets in each group; the x-axis indicates the groups. p values represent differences among groups as indicated.

Figure 4

Figure 4

Examples of AM expression of M1 and M2 polarization-related genes demonstrating progressive differences with the development of COPD. A. M1-related genes; and B. M2-related genes. Log2-transformed normalized expression levels for selected M1-related genes [C-X-C chemokine ligand 11 (CXCL11); C-X-C chemokine ligand 9 (CXCL9); guanylate binding protein (GBP 5)]; and M2-related genes [adenosine A3 receptor (ADORA3); matrix metalloprotease 2 (MMP2); and matrix metalloproteinase 7 (MMP7), are plotted for all healthy nonsmokers (n=24; green triangles), healthy smokers (n=32; orange triangles), and COPD smokers (n=12; blue triangles). p values are indicated. N.S., non-significant.

Figure 5

Figure 5

Principle component analysis (PCA) comparison of alveolar macrophage gene expression patterns of healthy smokers, healthy smokers and COPD smokers. A. PCA of all 26,959 probe sets expressed in AM of at least 20% of subjects. B. PCA of all 114 M1- and M2-related probe sets expressed in AM of at least 20% of subjects (see Tables I, II). In both analyses, all samples within each group were centered (left panels) or averaged and then the means were centered (right panels) in the three-dimensional space based on the expression pattern. In left panels, each circle represents an individual sample; in right panels, each circle represents an averaged sample for each group. Healthy nonsmokers, n=24, green; healthy smokers, n=34, orange; and COPD smokers, n=12, blue). The percentage contributions of the first three (left panels) or two (right panels) principal components (PC) to the observed variability between the groups are indicated.

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