Adherent Human Alveolar Macrophages Exhibit a Transient Pro-Inflammatory Profile That Confounds Responses to Innate Immune Stimulation

Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms ex vivo adherent AM are used immediately after isolation. Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM

Rac1 Deletion Causes Thymic Atrophy

The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation

Genome-wide comparison of MDM with freshly isolated and rested AM.

<p>(A) Hierarchical clustering of whole genome expression profiles showed greatest difference between MDM and freshly isolated AM, and closer clustering of rested AM to MDM. (B) Significant gene expression differences between rested AM and MDM assessed by gene ontology functional cluster analysis, showed greatest enrichment for upregulated expression in AM of genes associated with MHC class II antigen presentation pathways. (C) Mean relative gene expression levels are presented in MDM and AM for genes included in this cluster.</p

Transcription factor enrichment of highly upregulated genes in AM.

†<p>z-scores of >10 are considered to indicate highly significant over-representation of transcription factor binding sites within the analysed gene list.</p

Reduction of AM inflammatory gene expression with time in culture.

<p>(A) AM show a significant reduction in PTGS2 expression over time (p<0.0001, ANOVA). Data points represent technical replicates of measurements from one patient with DIP. (B) Selected gene expression changes identified by microarrays are validated by qPCR. Basal pro-inflammatory gene expression is reduced in AM which have been rested for 48 hours and upregulated following LPS stimulation. Bars represent mean ±SEM for at least 4 separate experiments (*denotes p<0.05, t-test).</p

Ontological associations of gene expression differences between AM and MDM.

*<p>Modified Fisher’s Exact Test.</p><p>Functional Annotation Clustering analysis by gene ontology classification of significant gene expression differences identified by transcriptional profiling of unstimulated AM (4 donors) and MDM (8 donors).</p

AM and MDM share common gene expression characteristics.

<p>(A) AM cultured for 24 hours exhibit similar appearances to MDM (differentiated for seven days) on light microscopy. (B) Mean relative gene expression levels of selected macrophage characteristic genes are comparable in AM (from four donors) and MDM (from eight donors). Many of these genes are also expressed in monocytes (from four donors), and to a lesser extent in DC (from five donors), but not in any of the cell lines. (C) Hierarchical clustering on the basis of macrophage signature genes also shows that AM and MDM transcriptional profiles are closely related, and segregate together with other myeloid cells, distinct from undifferentiated THP1, HeLa and SUPT1 cells and a standard reference RNA sample.</p

Inflammatory responses by freshly isolated AM.

<p>(A) Mean relative gene expression levels are presented in MDM and AM before and after 4–24 hour LPS stimulation, for the top 20 most upregulated genes following four hour LPS stimulation of MDM. (B) AM show extremely high baseline expression of pro-inflammatory genes which is not further upregulated following four hour LPS stimulation, however, after 24 hours in culture, basal inflammatory gene levels are reduced in AM, and LPS induced upregulation becomes evident. High basal pro-inflammatory cytokine levels which are not increased by LPS stimulation are also present in AM culture supernatants. Bars represent mean ± SEM for at least 4 separate experiments (*denotes p<0.05, t-test).</p

Differences in transcriptional profiles of AM and MDM.

<p>(A) Comparison of transcriptional profiles of AM and MDM shows extensive gene expression differences. (B) Functional annotation clustering of significant gene expression differences (>8-fold and p<0.05, t-test) shows that genes upregulated in AM are highly enriched for immune responses and cytokine activity, whereas downregulated genes show less significant enrichment for defense responses and extracellular processes (Modified Fisher’s Exact Test). (C) The three most significantly enriched gene ontology associations are shown for the top 30 most highly upregulated genes in AM.</p