Autoreactive CD4+ T lymphocytes play a vital role in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Since the discovery of T helper 17 cells, there is an ongoing debate whether T helper 1, T helper 17 or both subtypes of T lymphocytes are important for the initiation of autoimmune neuroinflammation. We examined peripheral blood CD4+ cells from patients with active and stable relapsing-remitting multiple sclerosis, and used mice with conditional deletion or over-expression of the transforming growth factor-beta inhibitor Smad7, to delineate the role of Smad7 in T cell differentiation and autoimmune neuroinflammation. We found that Smad7 is up-regulated in peripheral CD4+ cells from patients with multiple sclerosis during relapse but not remission, and that expression of Smad7 strongly correlates with T-bet, a transcription factor defining T helper 1 responses. Concordantly, mice with transgenic over-expression of Smad7 in T cells developed an enhanced disease course during experimental autoimmune encephalomyelitis, accompanied by elevated infiltration of inflammatory cells and T helper 1 responses in the central nervous system. On the contrary, mice with a T cell-specific deletion of Smad7 had reduced disease and central nervous system inflammation. Lack of Smad7 in T cells blunted T cell proliferation and T helper 1 responses in the periphery but left T helper 17 responses unaltered. Furthermore, frequencies of regulatory T cells were increased in the central nervous system of mice with a T cell-specific deletion and reduced in mice with a T cell-specific over-expression of Smad7. Downstream effects of transforming growth factor-beta on in vitro differentiation of naïve T cells to T helper 1, T helper 17 and regulatory T cell phenotypes were enhanced in T cells lacking Smad7. Finally, Smad7 was induced during T helper 1 differentiation and inhibited during T helper 17 differentiation. Taken together, the level of Smad7 in T cells determines T helper 1 polarization and regulates inflammatory cellular responses. Since a Smad7 deletion in T cells leads to immunosuppression, Smad7 may be a potential new therapeutic target in multiple sclerosis.

The action of type I interferons in the central nervous system (CNS) during autoimmunity is largely unknown. Here, we demonstrate elevated interferon beta concentrations in the CNS, but not blood, of mice with experimental autoimmune encephalomyelitis (EAE), a model for CNS autoimmunity. Furthermore, mice devoid of the broadly expressed type I IFN receptor (IFNAR) developed exacerbated clinical disease accompanied by a markedly higher inflammation, demyelination, and lethality without shifting the T helper 17 (Th17) or Th1 cell immune response. Whereas adoptive transfer of encephalitogenic T cells led to enhanced disease in Ifnar1(-/-) mice, newly created conditional mice with B or T lymphocyte-specific IFNAR ablation showed normal EAE. The engagement of IFNAR on neuroectodermal CNS cells had no protective effect. In contrast, absence of IFNAR on myeloid cells led to severe disease with an enhanced effector phase and increased lethality, indicating a distinct protective function of type I IFNs during autoimmune inflammation of the CNS.

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/EBPβ targets, and C/EBPβ deletion erased the long-term inscription of LPS-induced epigenetic marks and gene expression. Thus, short-term immune signaling can induce C/EBPβ-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.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants leading to functional impairment of the MeCP2 protein. Here, we used purified recombinant MeCP2e1 and MeCP2e2 protein variants fused to a TAT protein transduction domain (PTD) to evaluate their transduction ability into RTT patient-derived fibroblasts and the ability to carry out their cellular function. We then assessed their transduction ability and therapeutic effects in a RTT mouse model. In vitro, TAT-MeCP2e2-eGFP reversed the pathological hyperacetylation of histones H3K9 and H4K16, a hallmark of abolition of MeCP2 function. In vivo, intraperitoneal administration of TAT-MeCP2e1 and TAT-MeCP2e2 extended the lifespan of Mecp2-/y mice by >50%. This was accompanied by rescue of hippocampal CA2 neuron size in animals treated with TAT-MeCP2e1. Taken together, these findings provide a strong indication that recombinant TAT-MeCP2 can reach mouse brains following peripheral injection and can ameliorate the phenotype of RTT mouse models. Thus, our study serves as a first step in the development of a potentially novel RTT therapy.

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.

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.

Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are up-regulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such as the spinal cord, during experimental autoimmune encephalomyelitis (EAE). In this study, using competitive in vitro and in vivo assays, we show that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte-autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes/macrophages, but not in microglia, delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6Chi effector monocytes. Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance, and function.

Gut homeostasis and mucosal immune defense rely on the differential contributions of dendritic cells (DC) and macrophages. Here we show that colonic CX3CR1(+) mononuclear phagocytes are critical inducers of the innate response to Citrobacter rodentium infection. Specifically, the absence of IL-23 expression in macrophages or CD11b(+) DC results in the impairment of IL-22 production and in acute lethality. Highlighting immunopathology as a death cause, infected animals are rescued by the neutralization of IL-12 or IFNγ. Moreover, mice are also protected when the CD103(+) CD11b(-) DC compartment is rendered deficient for IL-12 production. We show that IL-12 production by colonic CD103(+) CD11b(-) DC is repressed by IL-23. Collectively, in addition to its role in inducing IL-22 production, macrophage-derived or CD103(-) CD11b(+) DC-derived IL-23 is required to negatively control the otherwise deleterious production of IL-12 by CD103(+) CD11b(-) DC. Impairment of this critical mononuclear phagocyte crosstalk results in the generation of IFNγ-producing former TH17 cells and fatal immunopathology.

Genome-encoded microRNAs (miRNAs) provide a posttranscriptional regulatory layer that controls the differentiation and function of various cellular systems, including hematopoietic cells. miR-142 is one of the most prevalently expressed miRNAs within the hematopoietic lineage. To address the in vivo functions of miR-142, we utilized a novel reporter and a loss-of-function mouse allele that we have recently generated. In this study, we show that miR-142 is broadly expressed in the adult hematopoietic system. Our data further reveal that miR-142 is critical for megakaryopoiesis. Genetic ablation of miR-142 caused impaired megakaryocyte maturation, inhibition of polyploidization, abnormal proplatelet formation, and thrombocytopenia. Finally, we characterized a network of miR-142-3p targets which collectively control actin filament homeostasis, thereby ensuring proper execution of actin-dependent proplatelet formation. Our study reveals a pivotal role for miR-142 activity in megakaryocyte maturation and function, and demonstrates a critical contribution of a single miRNA in orchestrating cytoskeletal dynamics and normal hemostasis.DOI: http://dx.doi.org/10.7554/eLife.01964.001.

Microglia seed the embryonic neuro-epithelium, expand and actively sculpt neuronal circuits in the developing central nervous system, but eventually adopt relative quiescence and ramified morphology in the adult. Here, we probed the impact of post-transcriptional control by microRNAs (miRNAs) on microglial performance during development and adulthood by generating mice lacking microglial Dicer expression at these distinct stages. Conditional Dicer ablation in adult microglia revealed that miRNAs were required to limit microglial responses to challenge. After peripheral endotoxin exposure, Dicer-deficient microglia expressed more pro-inflammatory cytokines than wild-type microglia and thereby compromised hippocampal neuronal functions. In contrast, prenatal Dicer ablation resulted in spontaneous microglia activation and revealed a role for Dicer in DNA repair and preservation of genome integrity. Accordingly, Dicer deficiency rendered otherwise radio-resistant microglia sensitive to gamma irradiation. Collectively, the differential impact of the Dicer ablation on microglia of the developing and adult brain highlights the changes these cells undergo with time.

Monocytes are circulating, short-lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C+ monocytes display developmental plasticity and are recruited to complement tissue-resident macrophages and dendritic cells on demand. Murine vascular Ly6C- monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA-seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C+ into Ly6C- monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre-established (poised) enhancers. Generation of Ly6C- monocytes involved induction of the transcription factor C/EBPβ and C/EBPβ-deficient mice lacked Ly6C- monocytes. Mechanistically, C/EBPβ bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor.

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.

CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor known to be involved in macrophage differentiation and function, steatohepatitis and liver fibrosis.

Classical dendritic cells (cDC) are specialized antigen-presenting cells mediating immunity and tolerance. cDC cell-lineage decisions are largely controlled by transcriptional factor regulatory cascades. Using an in vivo cell-specific targeting of Runx3 at various stages of DC lineage development we show that Runx3 is required for cell-identity, homeostasis and function of splenic Esam(hi) DC. Ablation of Runx3 in DC progenitors led to a substantial decrease in splenic CD4(+)/CD11b(+) DC. Combined chromatin immunoprecipitation sequencing and gene expression analysis of purified DC-subsets revealed that Runx3 is a key gene expression regulator that facilitates specification and homeostasis of CD11b(+)Esam(hi) DC. Mechanistically, loss of Runx3 alters Esam(hi) DC gene expression to a signature characteristic of WT Esam(low) DC. This transcriptional reprogramming caused a cellular change that diminished phagocytosis and hampered Runx3(-/-) Esam(hi) DC capacity to prime CD4(+) T cells, attesting to the significant role of Runx3 in specifying Esam(hi) DC identity and function.

Currently, there is little evidence for a notable role of the vertebrate microRNA (miRNA) system in the pathogenesis of RNA viruses. This is primarily attributed to the ease with which these viruses mutate to disrupt recognition and growth suppression by host miRNAs. Here we report that the haematopoietic-cell-specific miRNA miR-142-3p potently restricts the replication of the mosquito-borne North American eastern equine encephalitis virus in myeloid-lineage cells by binding to sites in the 3' non-translated region of its RNA genome. However, by limiting myeloid cell tropism and consequent innate immunity induction, this restriction directly promotes neurologic disease manifestations characteristic of eastern equine encephalitis virus infection in humans. Furthermore, the region containing the miR-142-3p binding sites is essential for efficient virus infection of mosquito vectors. We propose that RNA viruses can adapt to use antiviral properties of vertebrate miRNAs to limit replication in particular cell types and that this restriction can lead to exacerbation of disease severity.

An intriguing finding about the gene encoding methyl-CpG binding protein 2 (MeCP2) is that the loss-of-function mutations cause Rett syndrome and duplication (gain-of-function) of MECP2 leads to another neurological disorder termed MECP2 duplication syndrome. To ensure proper neurodevelopment, a precise regulation of MeCP2 expression is critical, and any gain or loss of MeCP2 over a narrow threshold level may lead to postnatal neurological impairment. To evaluate MeCP2 dosage effects, we generated Mecp2(WT_EGFP) transgenic (TG) mouse in which MeCP2 (endogenous plus TG) is mildly overexpressed (approximately 1.5×). The TG MeCP2(WT_EGFP) fusion protein is functionally active, as cross breeding of these mice with Mecp2 knockout mice led to alleviation of major phenotypes in the null mutant mice, including premature lethality. To characterize the Mecp2(WT_EGFP) mouse model, we performed an extensive battery of behavioral tests, which revealed that these mice manifest increased aggressiveness and higher pentylenetetrazole (PTZ)-induced seizure propensity. Evaluation of neuronal parameters revealed a reduction in the number of tertiary branching sites and increased spine density in Mecp2(WT_EGFP) transgenic (TG) neurons. Treatment of TG neurons with epileptogenic compound-PTZ led to a marked increase in amplitude and frequency of calcium spikes. Based on our ex vivo and in vivo data, we conclude that epileptic seizures are manifested as the first symptom when MeCP2 is mildly overexpressed in mice.

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/EBPβ deficiency by C/EBPε. Complementation was augmented by ectopic C/EBPβ expression and downstream activation of IGF1 that enhanced growth. Cebpe gene inactivation was accomplished only in the presence of complementing C/EBPβ, 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.

CCAAT/enhancer-binding protein beta (C/EBPβ) 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/EBPβ-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.