SummaryMacrophage activation is associated with profound transcriptional reprogramming. Although much progress has been made in the understanding of macrophage activation, polarization, and function, the transcriptional programs regulating these processes remain poorly characterized. We stimulated human macrophages with diverse activation signals, acquiring a data set of 299 macrophage transcriptomes. Analysis of this data set revealed a spectrum of macrophage activation states extending the current M1 versus M2-polarization model. Network analyses identified central transcriptional regulators associated with all macrophage activation complemented by regulators related to stimulus-specific programs. Applying these transcriptional programs to human alveolar macrophages from smokers and patients with chronic obstructive pulmonary disease (COPD) revealed an unexpected loss of inflammatory signatures in COPD patients. Finally, by integrating murine data from the ImmGen project we propose a refined, activation-independent core signature for human and murine macrophages. This resource serves as a framework for future research into regulation of macrophage activation in health and disease.

High Density Lipoprotein (HDL) mediates reverse cholesterol transport and it is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional repressor ATF3, as an HDL-inducible target gene in macrophages that down-regulates the expression of Toll-like receptor (TLR)-induced pro-inflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of novel HDL-based therapies.

Macrophages are dynamic cells integrating signals from their microenvironment to develop specific functional responses. Although, microarray-based transcriptional profiling has established transcriptional reprogramming as an important mechanism for signal integration and cell function of macrophages, current knowledge on transcriptional regulation of human macrophages is far from complete. To discover novel marker genes, an area of great need particularly in human macrophage biology but also to generate a much more thorough transcriptome of human M1- and M1-like macrophages, we performed RNA sequencing (RNA-seq) of human macrophages. Using this approach we can now provide a high-resolution transcriptome profile of human macrophages under classical (M1-like) and alternative (M2-like) polarization conditions and demonstrate a dynamic range exceeding observations obtained by previous technologies, resulting in a more comprehensive understanding of the transcriptome of human macrophages. Using this approach, we identify important gene clusters so far not appreciated by standard microarray techniques. In addition, we were able to detect differential promoter usage, alternative transcription start sites, and different coding sequences for 57 gene loci in human macrophages. Moreover, this approach led to the identification of novel M1-associated (CD120b, TLR2, SLAMF7) as well as M2-associated (CD1a, CD1b, CD93, CD226) cell surface markers. Taken together, these data support that high-resolution transcriptome profiling of human macrophages by RNA-seq leads to a better understanding of macrophage function and will form the basis for a better characterization of macrophages in human health and disease.

Regulatory T (Treg) cells are essential for self-tolerance and immune homeostasis. Lack of effector T cell (Teff) function and gain of suppressive activity by Treg are dependent on the transcriptional program induced by Foxp3. Here we report repression of SATB1, a genome organizer regulating chromatin structure and gene expression, as crucial for Treg phenotype and function. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly through induction of microRNAs that bound the SATB1 3′UTR. Release of SATB1 from Foxp3 control in Treg caused loss of suppressive function, establishment of transcriptional Teff programs and induction of Teff cytokines. These data support that inhibition of SATB1-mediated modulation of global chromatin remodelling is pivotal for maintaining Treg functionality.

SummaryHuman in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.