Transcriptional and functional diversity of human macrophage repolarization

Abstract

Background: Macrophage plasticity allows cells to adopt different phenotypes, a property with important implications in disorders such as cystic fibrosis (CF) and asthma.

Objective: We sought to examine the transcriptional and functional significance of macrophage repolarization from an M1 to an M2 phenotype and assess the role of a common human genetic disorder (CF) and a prototypical allergic disease (asthma) in this transformation.

Methods: Monocyte-derived macrophages were collected from healthy subjects and patients with CF and polarized to an M2 state by using IL-4, IL-10, glucocorticoids, apoptotic PMNs, or azithromycin. We performed transcriptional profiling and pathway analysis for each stimulus. We assessed the ability of M2-repolarized macrophages to respond to LPS rechallenge and clear apoptotic neutrophils and used murine models to determine conserved functional responses to IL-4 and IL-10. We investigated whether M2 signatures were associated with alveolar macrophage phenotypes in asthmatic patients.

Results: We found that macrophages exhibit highly diverse responses to distinct M2-polarizing stimuli. Specifically, IL-10 activated proinflammatory pathways and abrogated LPS tolerance, allowing rapid restoration of LPS responsiveness. In contrast, IL-4 enhanced LPS tolerance, dampening proinflammatory responses after repeat LPS challenge. A common theme observed across all M2 stimuli was suppression of interferon-associated pathways. We found that CF macrophages had intact reparative and transcriptional responses, suggesting that macrophage contributions to CF-related lung disease are primarily shaped by their environment. Finally, we leveraged in vitro-derived signatures to show that allergen provocation induces distinct M2 state transcriptional patterns in alveolar macrophages.

Conclusion: Our findings highlight the diversity of macrophage polarization, attribute functional consequences to different M2 stimuli, and provide a framework to phenotype macrophages in disease states.

Keywords: Macrophage; asthma; cystic fibrosis; efferocytosis; microarray; pathway; polarization; tolerance.

Figure 1.. Overview of experimental design.

(A) Blood monocytes from 6 non-CF and 6 CF donors were cultured with M-CSF over 7 days to produce MDMs. Cells were stimulated with E. coli LPS for 24h and then repolarized with IL-10, IL-4, azithromycin, methylprednisolone, apoptotic PMNs, or control media for 24 h. Cells were then processed for gene expression and functional assays. (B) Hierarchical cluster analysis of a total of 5,317 differentially expressed genes in non-CF and CF subjects across the five M2 stimuli. Note the distinct transcriptional pattern associated with each exposure, and the overall similar expression profile between non-CF and CF subjects. An FDR < 0.01 cutoff was used to designate significant differential expression in each M2 exposure relative to baseline (no treatment). (C) Principal Component Analysis. To simplify visualization of the 72 experiments, differentially expressed transcriptional profiles were averaged in CF (n = 6) and non-CF (n = 6) subjects for each M2 stimulus. Each orthogonal axis captures a percentage of the total gene expression variability, with ~80% of the variance captured by the three axes. Note that the non-CF and CF samples segregate based on M2 stimuli, implying that the primary determinant of separation between groups is exposure, not genotype

Figure 2.. Visual summary of pathway analysis.

(A) GSEA of the transcriptional response to M2-polarizing stimuli identified many differentially up and down-regulated pathways. Network analysis was used to group these pathways into seven larger aggregates known as modules. Note the divergent enrichment pattern across conditions, although common themes such as suppression of interferon-associated pathways are also revealed from this analysis. Complete list of enriched gene sets is provided in Supplemental Table E3. (B) Network-based visualization of the GSEA of MDM response to IL-10 treatment illustrates the emergence of modules comprised of interconnected pathways (shown as spheres). Unlike other M2 stimuli, exposure to IL-10 activated multiple immune-related programs, several of which are listed.

Figure 3.. CD14 is differentially regulated by IL-4 and IL-10.

Expression of the LPS-binding protein and TLR4 co-receptor, CD14 was significantly suppressed by all M2-stimuli with the exception of IL-10, which significantly increased its expression as shown by (A) mRNA from gene expression arrays, and (B) protein as measured by flow cytometry. (C) CD14 staining (yellow) on the cell membrane of non-CF MDMs as imaged using Image Stream. MFI=median fluorescent intensity. BF= bright field. (Mean ± SEM; n=6/group).

Figure 4.. Consequences of M2 repolarization on LPS rechallenge.

Monocyte-derived macrophages from 6 healthy donors were stimulated with LPS (+) followed by M2 stimuli or control medium and then rechallenged with LPS. Additional control samples did not receive the first LPS challenge (-/Medium/+). Each donor’s samples were normalized to HPRT and expressed as fold change over their respective non-LPS treated samples. (A-F) For II1b, II6, II8, CxcI1, CxcI2 but not Tnfa, IL-4 exposure decreased the ability of the macrophage to produce pro-inflammatory cytokines, and IL-10 reversed LPS tolerance. (Mean ± SEM; n=6/group). *P-value <0.05.

Figure 5.. Murine macrophage inflammatory response to LPS rechallenge is modified by IL-4 and IL-10.

Stat6^−/−, II10r^−/−, and WT bone marrow-derived macrophages were stimulated with LPS followed by IL-4, IL-10, or control media and then rechallenged with LPS. Samples were normalized to HPRT and expressed as fold change over their respective control media-treated samples. (A) Experiments performed in BALB-C wildtype (WT) and Stat6^−/− BMDM demonstrating a Stat6-dependent suppression of LPS-rechallenge by IL-4. (I) Experiments performed in C57BL/6 WT and II10r^−/− BMDM demonstrating an IL-10r-dependent augmentation of LPS-rechallenge by IL-10. Each condition was performed in triplicate and 2 experiment replicates performed. *P-value < 0.05.

Figure 6.. M2 stimuli have varying ability to promote macrophage efferocytosis and are independent of functional CFTR.

(A) Representative gating strategy to quantify efferocytosis of CD14+ macrophages using CFSE⁺ PMNs. The CFSE⁺ gate was drawn using control macrophages incubated with unlabeled PMNs. The percentage of CD14⁺ macrophages positive for ingested apoptotic PMNs (% CFSE⁺) and quantity of ingested apoptotic PMNs (CFSE MFI) was determined across stimuli. (B) Fold change in percentage of macrophages positive for ingested apoptotic PMNs (PMN) relative to their respective media-only controls. (C) Fold change in CFSE MFI in macrophages relative to their respective media-only controls. *P-value < 0.05 M2 stimuli vs. media control. AZM=azithromycin, MP=methylprednisolone, PMN=apoptotic PMNs, MFI=Mean fluorescence intensity. (Mean ± SEM; n=6/group).