Long-term epigenetic reprogramming of innate immune cells in response to microbes, also termed "trained immunity," causes prolonged altered cellular functionality to protect from secondary infections. Here, we investigated whether sterile triggers of inflammation induce trained immunity and thereby influence innate immune responses. Western diet (WD) feeding of Ldlr mice induced systemic inflammation, which was undetectable in serum soon after mice were shifted back to a chow diet (CD). In contrast, myeloid cell responses toward innate stimuli remained broadly augmented. WD-induced transcriptomic and epigenomic reprogramming of myeloid progenitor cells led to increased proliferation and enhanced innate immune responses. Quantitative trait locus (QTL) analysis in human monocytes trained with oxidized low-density lipoprotein (oxLDL) and stimulated with lipopolysaccharide (LPS) suggested inflammasome-mediated trained immunity. Consistently, Nlrp3/Ldlr mice lacked WD-induced systemic inflammation, myeloid progenitor proliferation, and reprogramming. Hence, NLRP3 mediates trained immunity following WD and could thereby mediate the potentially deleterious effects of trained immunity in inflammatory diseases.

Resident macrophages support embryonic development and tissue homeostasis and repair. The mechanisms that control their differentiation remain unclear. We report here that Erythro-Myeloid Progenitors generate pre-macrophages (pMacs) that simultaneously colonize the whole embryo from embryonic day (E)9.5 in a chemokine-receptor dependent manner. The core macrophage program initiated in pMacs is rapidly diversified as expression of transcriptional regulators becomes tissue-specific in early macrophages. This process appears essential for macrophage specification and maintenance, as inactivation of Id3 impairs the development of liver macrophages and results in selective Kupffer cell deficiency in adults. We propose that macrophage differentiation is an integral part of organogenesis as colonization of organ anlagen by pMacs is followed by their specification into tissue macrophages, hereby generating the macrophage diversity observed in postnatal tissues.

Dendritic cells (DC) are professional antigen-presenting cells that orchestrate immune responses. The human DC population comprises two main functionally specialized lineages, whose origins and differentiation pathways remain incompletely defined. Here, we combine two high-dimensional technologies-single-cell messenger RNA sequencing (scmRNAseq) and cytometry by time-of-flight (CyTOF)-to identify human blood CD123CD33CD45RA DC precursors (pre-DC). Pre-DC share surface markers with plasmacytoid DC (pDC) but have distinct functional properties that were previously attributed to pDC. Tracing the differentiation of DC from the bone marrow to the peripheral blood revealed that the pre-DC compartment contains distinct lineage-committed subpopulations, including one early uncommitted CD123 pre-DC subset and two CD45RACD123 lineage-committed subsets exhibiting functional differences. The discovery of multiple committed pre-DC populations opens promising new avenues for the therapeutic exploitation of DC subset-specific targeting.

Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer. We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1(-/-) mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity. Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes. Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses.

Regulatory networks for differentiation and pluripotency in embryonic stem (ES) cells have long been suggested to be mutually exclusive. However, with the identification of many new components of these networks ranging from epigenetic, transcriptional, and translational to even post-translational mechanisms, the cellular states of pluripotency and early differentiation might not be strictly bi-modal, but differentiating stem cells appear to go through phases of simultaneous expression of stemness and differentiation genes. Translational regulators such as RNA binding proteins (RBPs) and micro RNAs (miRNAs) might be prime candidates for guiding a cell from pluripotency to differentiation. Using Trim71, one of two members of the Tripartite motif (Trim) protein family with RNA binding activity expressed in murine ES cells, we demonstrate that Trim71 is not involved in regulatory networks of pluripotency but regulates neural differentiation. Loss of Trim71 in mES cells leaves stemness and self-maintenance of these cells intact, but many genes required for neural development are up-regulated at the same time. Concordantly, Trim71−/− mES show increased neural marker expression following treatment with retinoic acid. Our findings strongly suggest that Trim71 keeps priming steps of differentiation in check, which do not pre-require a loss of the pluripotency network in ES cells.

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.

Differentiation of inflammatory macrophages from monocytes is characterized by an orderly integration of epigenetic and transcriptional regulatory mechanisms guided by lineage-determining transcription factors such as PU.1. Further activation of macrophages leads to a stimulus- or microenvironment-specific signal integration with subsequent transcriptional control established by the action of tissue- or signal-associated transcription factors. Here, we assess four histone modifications during human macrophage activation and integrate this information with the gene expression data from 28 different macrophage activation conditions in combination with GM-CSF. Bioinformatically, for inflammatory macrophages we define a unique network of transcriptional and epigenetic regulators (TRs), which was characterized by accessible promoters independent of the activation signal. In contrast to the general accessibility of promoters of TRs, mRNA expression of central TRs belonging to the TR network displayed stimulus-specific expression patterns, indicating a second level of transcriptional regulation beyond epigenetic chromatin changes. In contrast, stringent integration of epigenetic and transcriptional regulation was observed in networks of TRs established from somatic tissues and tissue macrophages. In these networks, clusters of TRs with permissive histone marks were associated with high gene expression whereas clusters with repressive chromatin marks were associated with absent gene expression. Collectively, these results support that macrophage activation during inflammation in contrast to lineage determination is mainly regulated transcriptionally by a pre-defined TR network.

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