C/EBPß-induced lymphoid-to-myeloid transdifferentiation emulates granulocyte-monocyte progenitor biology.

CCAAT/enhancer-binding protein beta (C/EBPß) induces primary v-Abl immortalized mouse B cells to transdifferentiate (BT, B cell transdifferentiation) into granulocyte-macrophage progenitor-like cells (GMPBTs). GMPBTs maintain cytokine-independent self-renewal, lineage choice, and multilineage differentiation. Single-cell transcriptomics demonstrated that GMPBTs comprise a continuum of myelomonopoietic differentiation states that seamlessly fit into state-to-fate maps of normal granulocyte-macrophage progenitors (GMPs). Inactivating v-Abl kinase revealed the dependence on activated CSF2-JAK2-STAT5 signaling. Deleting IRF8 diminished monopoiesis and enhanced granulopoiesis while removing C/EBPß-abrogated self-renewal and granulopoiesis but permitted macrophage differentiation. The GMPBT culture system is easily scalable to explore the basics of GMP biology and lineage commitment and largely reduces ethically and legislatively debatable, labor-intensive, and costly animal experiments.

Carm1-arginine methylation of the transcription factor C/EBPα regulates transdifferentiation velocity.

Here, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPαR35A) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPαR35A, initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme's perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell's differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.

Immune cell C/EBPß deficiency is associated with hepatic mononuclear defects and spontaneous hepatitis but not steatohepatitis induced liver fibrosis.

BACKGROUND: CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor known to be involved in macrophage differentiation and function, steatohepatitis and liver fibrosis. METHODS: Immune restricted C/EBPß deficient and control mice were investigated in steady-state and in the CDA-HFD steatohepatitis model. Mice were assessed for weight change, liver biochemical profile, histology and hepatic phagocytes composition. RESULTS: Flow cytometry analysis of hepatic nonparenchymal cells revealed reduced numbers of hepatic monocytes and Kupffer cells and an increase in hepatic MHC class II positive myeloid cells in immune cells restricted C/EBPß deficient mice. Immune-restricted C/EBPß deficiency resulted in decreased weight gain and appearance of mild spontaneous liver inflammation. Nevertheless, In the CDA-HFD steatohepatitis model, immune restricted C/EBPß deficient and proficient mice exhibit similar grade of hepatic steatosis, liver enzymes levels and fibrosis stage. CONCLUSIONS: Immune-restricted C/EBPß deficiency leads to significant alteration in hepatic mononuclear phagocytes composition associated with spontaneous mild hepatitis. Steatohepatitis associated fibrosis is not dependent on C/EBPß expression by immune cells.

C/EBPß regulates lipid metabolism and Pparg isoform 2 expression in alveolar macrophages.

Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant lipoproteins within the lung alveoli. Alveolar macrophages (AMs) are crucial for surfactant clearance, and their differentiation depends on colony-stimulating factor 2 (CSF2), which regulates the establishment of an AM-characteristic gene regulatory network. Here, we report that the transcription factor CCAAT/enhancer binding protein ß (C/EBPß) is essential for the development of the AM identity, as demonstrated by transcriptome and chromatin accessibility analysis. Furthermore, C/EBPß-deficient AMs showed severe defects in proliferation, phagocytosis, and lipid metabolism, collectively resulting in a PAP-like syndrome. Mechanistically, the long C/EBPß protein variants LAP* and LAP together with CSF2 signaling induced the expression of Pparg isoform 2 but not Pparg isoform 1, a molecular regulatory mechanism that was also observed in other CSF2-primed macrophages. These results uncover C/EBPß as a key regulator of AM cell fate and shed light on the molecular networks controlling lipid metabolism in macrophages.

Tongue immune compartment analysis reveals spatial macrophage heterogeneity.

The tongue is a unique muscular organ situated in the oral cavity where it is involved in taste sensation, mastication, and articulation. As a barrier organ, which is constantly exposed to environmental pathogens, the tongue is expected to host an immune cell network ensuring local immune defence. However, the composition and the transcriptional landscape of the tongue immune system are currently not completely defined. Here, we characterised the tissue-resident immune compartment of the murine tongue during development, health and disease, combining single-cell RNA-sequencing with in situ immunophenotyping. We identified distinct local immune cell populations and described two specific subsets of tongue-resident macrophages occupying discrete anatomical niches. Cx3cr1+ macrophages were located specifically in the highly innervated lamina propria beneath the tongue epidermis and at times in close proximity to fungiform papillae. Folr2+ macrophages were detected in deeper muscular tissue. In silico analysis indicated that the two macrophage subsets originate from a common proliferative precursor during early postnatal development and responded differently to systemic LPS in vivo. Our description of the under-investigated tongue immune system sets a starting point to facilitate research on tongue immune-physiology and pathology including cancer and taste disorders.

CSF2-dependent monocyte education in the pathogenesis of ANCA-induced glomerulonephritis.

OBJECTIVES: Myeloid cell activation by antineutrophil cytoplasmic antibody (ANCA) is pivotal for necrotising vasculitis, including necrotising crescentic glomerulonephritis (NCGN). In contrast to neutrophils, the contribution of classical monocyte (CM) and non-classical monocyte (NCM) remains poorly defined. We tested the hypothesis that CMs contribute to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) and that colony-stimulating factor-2 (CSF2, granulocyte-macrophage colony-stimulating factor (GM-CSF)) is an important monocyte-directed disease modifier. METHODS: Myeloperoxidase (MPO)-immunised MPO-/- mice were transplanted with haematopoietic cells from wild-type (WT) mice, C-C chemokine receptor 2 (CCR2)-/- mice to abrogate CM, or transcription factor CCAAT-enhancer-binding protein beta (C/EBPß)-/- mice to reduce NCM, respectively. Monocytes were stimulated with CSF2, and CSF2 receptor subunit beta (CSF2rb)-deficient mice were used. Urinary monocytes and CSF2 were quantified and kidney Csf2 expression was analysed. CSF2-blocking antibody was used in the nephrotoxic nephritis (NTN) model. RESULTS: Compared with WT mice, CCR2-/- chimeric mice showed reduced circulating CM and were protected from NCGN. C/EBPß-/- chimeric mice lacked NCM but developed NCGN similar to WT chimeric mice. Kidney and urinary CSF2 were upregulated in AAV mice. CSF2 increased the ability of ANCA-stimulated monocytes to generate interleukin-1ß and to promote TH17 effector cell polarisation. CSF2rb-/- chimeric mice harboured reduced numbers of kidney TH17 cells and were protected from NCGN. CSF2 neutralisation reduced renal damage in the NTN model. Finally, patients with active AAV displayed increased urinary CM numbers, CSF2 levels and expression of GM-CSF in infiltrating renal cells. CONCLUSIONS: CMs but not NCMs are important for inducing kidney damage in AAV. CSF2 is a crucial pathological factor by modulating monocyte proinflammatory functions and thereby TH17 cell polarisation.

A Universal Peptide Matrix Interactomics Approach to Disclose Motif-Dependent Protein Binding.

Protein-protein interactions mediated by intrinsically disordered regions are often based on short linear motifs (SLiMs). SLiMs are implicated in signal transduction and gene regulation yet remain technically laborious and notoriously challenging to study. Here, we present an optimized method for a protein interaction screen on a peptide matrix (PRISMA) in combination with quantitative MS. The protocol was benchmarked with previously described SLiM-based protein-protein interactions using peptides derived from EGFR, SOS1, GLUT1, and CEBPB and extended to map binding partners of kinase activation loops. The detailed protocol provides practical considerations for setting up a PRISMA screen and subsequently implementing PRISMA on a liquid-handling robotic platform as a cost-effective high-throughput method. Optimized PRISMA can be universally applied to systematically study SLiM-based interactions and associated post-translational modifications or mutations to advance our understanding of the largely uncharacterized interactomes of intrinsically disordered protein regions.

PRISMA and BioID disclose a motifs-based interactome of the intrinsically disordered transcription factor C/EBPα.

C/EBPα represents a paradigm intrinsically disordered transcription factor containing short linear motifs and post-translational modifications (PTM). Unraveling C/EBPα protein interaction networks is a prerequisite for understanding the multi-modal functions of C/EBPα in hematopoiesis and leukemia. Here, we combined arrayed peptide matrix screening (PRISMA) with BioID to generate an in vivo validated and isoform specific interaction map of C/EBPα. The myeloid C/EBPα interactome comprises promiscuous and PTM-regulated interactions with protein machineries involved in gene expression, epigenetics, genome organization, DNA replication, RNA processing, and nuclear transport. C/EBPα interaction hotspots coincide with homologous conserved regions of the C/EBP family that also score as molecular recognition features. PTMs alter the interaction spectrum of C/EBP-motifs to configure a multi-valent transcription factor hub that interacts with multiple co-regulatory components, including BAF/SWI-SNF or Mediator complexes. Combining PRISMA and BioID is a powerful strategy to systematically explore the PTM-regulated interactomes of intrinsically disordered transcription factors.

Myeloid transformation by MLL-ENL depends strictly on C/EBP.

Chromosomal rearrangements of the mixed-lineage leukemia gene MLL1 are the hallmark of infant acute leukemia. The granulocyte-macrophage progenitor state forms the epigenetic basis for myelomonocytic leukemia stemness and transformation by MLL-type oncoproteins. Previously, it was shown that the establishment of murine myelomonocytic MLL-ENL transformation, but not its maintenance, depends on the transcription factor C/EBPα, suggesting an epigenetic hit-and-run mechanism of MLL-driven oncogenesis. Here, we demonstrate that compound deletion of Cebpa/Cebpb almost entirely abrogated the growth and survival of MLL-ENL-transformed cells. Rare, slow-growing, and apoptosis-prone MLL-ENL-transformed escapees were recovered from compound Cebpa/Cebpb deletions. The escapees were uniformly characterized by high expression of the resident Cebpe gene, suggesting inferior functional compensation of C/EBPα/C/EBPß deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPß expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPß, but not in its absence, confirming the Cebpe dependency of the Cebpa/Cebpb double knockouts. Our data show that MLL-transformed myeloid cells are dependent on C/EBPs during the initiation and maintenance of transformation.

The transcription factor C/EBPß orchestrates dendritic cell maturation and functionality under homeostatic and malignant conditions.

Dendritic cell (DC) maturation is a prerequisite for the induction of adaptive immune responses against pathogens and cancer. Transcription factor (TF) networks control differential aspects of early DC progenitor versus late-stage DC cell fate decisions. Here, we identified the TF C/EBPß as a key regulator for DC maturation and immunogenic functionality under homeostatic and lymphoma-transformed conditions. Upon cell-specific deletion of C/EBPß in CD11c+MHCIIhi DCs, gene expression profiles of splenic C/EBPß-/- DCs showed a down-regulation of E2F cell cycle target genes and associated proliferation signaling pathways, whereas maturation signatures were enriched. Total splenic DC cell numbers were modestly increased but differentiation into cDC1 and cDC2 subsets were unaltered. The splenic CD11c+MHCIIhiCD64+ DC compartment was also increased, suggesting that C/EBPß deficiency favors the expansion of monocytic-derived DCs. Expression of C/EBPß could be mimicked in LAP/LAP* isoform knockin DCs, whereas the short isoform LIP supported a differentiation program similar to deletion of the full-length TF. In accordance with E2F1 being a negative regulator of DC maturation, C/EBPß-/- bone marrow-derived DCs matured much faster enabling them to activate and polarize T cells stronger. In contrast to a homeostatic condition, lymphoma-exposed DCs exhibited an up-regulation of the E2F transcriptional pathways and an impaired maturation. Pharmacological blockade of C/EBPß/mTOR signaling in human DCs abrogated their protumorigenic function in primary B cell lymphoma cocultures. Thus, C/EBPß plays a unique role in DC maturation and immunostimulatory functionality and emerges as a key factor of the tumor microenvironment that promotes lymphomagenesis.

Author Correction: Cxcl10+ monocytes define a pathogenic subset in the central nervous system during autoimmune neuroinflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Cxcl10+ monocytes define a pathogenic subset in the central nervous system during autoimmune neuroinflammation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cxcl10+ monocytes define a pathogenic subset in the central nervous system during autoimmune neuroinflammation.

Multiple sclerosis (MS) is characterized by pathological inflammation that results from the recruitment of lymphoid and myeloid immune cells from the blood into the brain. Due to subset heterogeneity, defining the functional roles of the various cell subsets in acute and chronic stages of MS has been challenging. Here, we used index and transcriptional single-cell sorting to characterize the mononuclear phagocytes that infiltrate the central nervous system from the periphery in mice with experimentally induced autoimmune encephalomyelitis, a model of MS. We identified eight monocyte and three dendritic cell subsets at acute and chronic disease stages in which the defined transcriptional programs pointed toward distinct functions. Monocyte-specific cell ablation identified Cxcl10+ and Saa3+ monocytic subsets with a pathogenic potential. Transfer experiments with different monocyte and precursor subsets indicated that these Cxcl10+ and Saa3+ pathogenic cells were not derived from Ly6C+ monocytes but from early myeloid cell progenitors. These results suggest that blocking specific pathogenic monocytic subsets, including Cxcl10+ and Saa3+ monocytes, could be used for targeted therapeutic interventions.

C/EBPß-Dependent Epigenetic Memory Induces Trained Immunity in Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) maintain life-long production of immune cells and can directly respond to infection, but sustained effects on the immune response remain unclear. We show that acute immune stimulation with lipopolysaccharide (LPS) induced only transient changes in HSC abundance, composition, progeny, and gene expression, but persistent alterations in accessibility of specific myeloid lineage enhancers occurred, which increased responsiveness of associated immune genes to secondary stimulation. Functionally, this was associated with increased myelopoiesis of pre-exposed HSCs and improved innate immunity against the gram-negative bacterium P. aeruginosa. The accessible myeloid enhancers were enriched for C/EBPß targets, and C/EBPß deletion erased the long-term inscription of LPS-induced epigenetic marks and gene expression. Thus, short-term immune signaling can induce C/EBPß-dependent chromatin accessibility, resulting in HSC-trained immunity, during secondary infection. This establishes a mechanism for how infection history can be epigenetically inscribed in HSCs as an integral memory function of innate immunity.

PRISMA: Protein Interaction Screen on Peptide Matrix Reveals Interaction Footprints and Modifications- Dependent Interactome of Intrinsically Disordered C/EBPß.

CCAAT enhancer-binding protein beta (C/EBPß) is a pioneer transcription factor that specifies cell differentiation. C/EBPß is intrinsically unstructured, a molecular feature common to many proteins involved in signal processing and epigenetics. The structure of C/EBPß differs depending on alternative translation initiation and multiple post-translational modifications (PTM). Mutation of distinct PTM sites in C/EBPß alters protein interactions and cell differentiation, suggesting that a C/EBPß PTM indexing code determines epigenetic outcomes. Herein, we systematically explored the interactome of C/EBPß using an array technique based on spot-synthesized C/EBPß-derived linear tiling peptides with and without PTM, combined with mass spectrometric proteomic analysis of protein interactions. We identified interaction footprints of ∼1,300 proteins in nuclear extracts, many with chromatin modifying, chromatin remodeling, and RNA processing functions. The results suggest that C/EBPß acts as a multi-tasking molecular switchboard, integrating signal-dependent modifications and structural plasticity to orchestrate interactions with numerous protein complexes directing cell fate and function.

A C/EBPα-Wnt connection in gut homeostasis and carcinogenesis.

We explored the connection between C/EBPα (CCAAT/enhancer-binding protein α) and Wnt signaling in gut homeostasis and carcinogenesis. C/EBPα was expressed in human and murine intestinal epithelia in the transit-amplifying region of the crypts and was absent in intestinal stem cells and Paneth cells with activated Wnt signaling. In human colorectal cancer and murine APCMin/+ polyps, C/EBPα was absent in the nuclear ß-catenin-positive tumor cells. In chemically induced intestinal carcinogenesis, C/EBPα KO in murine gut epithelia increased tumor volume. C/EBPα deletion extended the S-phase cell zone in intestinal organoids and activated typical proliferation gene expression signatures, including that of Wnt target genes. Genetic activation of ß-catenin in organoids attenuated C/EBPα expression, and ectopic C/EBPα expression in HCT116 cells abrogated proliferation. C/EBPα expression accompanied differentiation of the colon cancer cell line Caco-2, whereas ß-catenin stabilization suppressed C/EBPα. These data suggest homeostatic and oncogenic suppressor functions of C/EBPα in the gut by restricting Wnt signaling.

The C/EBPß LIP isoform rescues loss of C/EBPß function in the mouse.

The transcription factor C/EBPß regulates hematopoiesis, bone, liver, fat, and skin homeostasis, and female reproduction. C/EBPß protein expression from its single transcript occurs by alternative in-frame translation initiation at consecutive start sites to generate three isoforms, two long (LAP*, LAP) and one truncated (LIP), with the same C-terminal bZip dimerization domain. The long C/EBPß isoforms are considered gene activators, whereas the LIP isoform reportedly acts as a dominant-negative repressor. Here, we tested the putative repressor functions of the C/EBPß LIP isoform in mice by comparing monoallelic WT or LIP knockin mice with Cebpb knockout mice, in combination with monoallelic Cebpa mice. The C/EBPß LIP isoform was sufficient to function in coordination with C/EBPα in murine development, adipose tissue and sebocyte differentiation, and female fertility. Thus, the C/EBPß LIP isoform likely has more physiological functions than its currently known role as a dominant-negative inhibitor, which are more complex than anticipated.