Physical and functional interaction among Irf8 enhancers during dendritic cell differentiation.

The production of type 1 conventional dendritic cells (cDC1s) requires high expression of the transcription factor IRF8. Three enhancers at the Irf8 3' region function in a differentiation stage-specific manner. However, whether and how these enhancers interact physically and functionally remains unclear. Here, we show that the Irf8 3' enhancers directly interact with each other and contact the Irf8 gene body during cDC1 differentiation. The +56 kb enhancer, which functions from multipotent progenitor stages, activates the other 3' enhancers through an IRF8-dependent transcription factor program, that is, in trans. Then, the +32 kb enhancer, which operates in cDC1-committed cells, reversely acts in cis on the other 3' enhancers to maintain the high expression of Irf8. Indeed, mice with compound heterozygous deletion of the +56 and +32 kb enhancers are unable to generate cDC1s. These results illustrate how multiple enhancers cooperate to induce a lineage-determining transcription factor gene during cell differentiation.

A single-cell genomic strategy for alternative transcript start sites identification.

Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-ß stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-ß stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.

Type I IFN Derived from Ly6Chi Monocytes Suppresses Type 2 Inflammation in a Murine Model of Atopic Dermatitis.

The roles of innate immune cells, including eosinophils, basophils, and group 2 innate lymphoid cells, in atopic dermatitis (AD) have been well-documented, whereas that of monocytes, another component of the innate immunity, remains rather poorly understood, thus necessitating the topic of this study. In addition, cytokines and cellular pathways needed for the resolution of type 2 inflammation in AD need further investigation. Using a murine AD model, we report here that (i) Ly6Chi monocytes were rapidly recruited to the AD lesion in a CCR2-dependent manner, blockade of which exacerbated AD; (ii) type I IFN production is profoundly involved in this suppression because the blockade of it by genetic depletion or antibody neutralization exacerbated AD; and (iii) Ly6Chi monocytes operate through the production of type I IFN because Ly6Chi monocytes from Irf7-null mice, which lack type I IFN production, failed to rescue Ccr2-/- mice from severe AD upon adoptive transfer. In addition, in vitro studies demonstrated type I IFN suppressed basophil expansion from bone marrow progenitor cells and survival of mature basophils. Collectively, our work suggests that Ly6Chi monocytes are the first and dominant inflammatory cells reaching AD lesions that negatively regulate type 2 inflammation through the production of type I IFN.

BRD4 directs mitotic cell division by inhibiting DNA damage.

BRD4 binds to acetylated histones to regulate transcription and drive cancer cell proliferation. However, the role of BRD4 in normal cell growth remains to be elucidated. Here we investigated the question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. We suggest that BRD4 epigenetically marks those genes and serves as a master regulator of normal cell growth.

IRF8 configures enhancer landscape in postnatal microglia and directs microglia specific transcriptional programs.

Microglia are innate immune cells in the brain. Transcription factor IRF8 is highly expressed in microglia. However, its role in postnatal microglia development and the mechanism of action is unknown. We demonstrate here that IRF8 binds to enhancer regions of postnatal microglia in a stepwise fashion reaching a maximum after day 14, which coincided with the initiation of microglia function. Constitutive Irf8 deletion led to the loss of microglia identity gene expression and aberrant induction of Alzheimer's disease and neurodegeneration associated genes. Conditional Irf8 deletion in adult microglia showed similar transcriptome profiles, revealing the requirement of continuous IRF8 expression. Additional genome-wide analyses showed IRF8 is critical for setting microglia-specific chromatin accessibility and DNA methylation patterns. Lastly, in the 5xFAD mouse AD model, Irf8 deletion lessened the formation and spread of amyloidß plaques, thereby reducing neuronal loss. Together, IRF8 sets the microglia epigenome landscape, required for eliciting microglia identity and function.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism through which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in the injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3-/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8-/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8-/- bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism through which migrating macrophages attract astrocytes and affect the pathophysiology and outcome after SCI.

Macrophages play a leading role in determining the direction of astrocytic migration in spinal cord injury via ADP-P2Y1R axis.

After spinal cord injury (SCI), inflammatory cells such as macrophages infiltrate the injured area, and astrocytes migrate, forming a glial scar around macrophages. The glial scar inhibits axonal regeneration, resulting in significant permanent disability. However, the mechanism by which glial scar-forming astrocytes migrate to the injury site has not been clarified. Here we show that migrating macrophages attract reactive astrocytes toward the center of the lesion after SCI. Chimeric mice with bone marrow lacking IRF8, which controls macrophage centripetal migration after SCI, showed widely scattered macrophages in injured spinal cord with the formation of a huge glial scar around the macrophages. To determine whether astrocytes or macrophages play a leading role in determining the directions of migration, we generated chimeric mice with reactive astrocyte-specific Socs3 -/- mice, which showed enhanced astrocyte migration, and bone marrow from IRF8 -/- mice. In this mouse model, macrophages were widely scattered, and a huge glial scar was formed around the macrophages as in wild-type mice that were transplanted with IRF8 -/ bone marrow. In addition, we revealed that macrophage-secreted ATP-derived ADP attracts astrocytes via the P2Y1 receptor. Our findings revealed a mechanism in which migrating macrophages attracted astrocytes and affected the pathophysiology and outcome after SCI.

Immature functional development of lumbar locomotor networks in adult Irf8-/- mice.

To date, research on the role of the brainstem and spinal cord in motor behavior has relied on in vitro preparations of the neonatal rodent spinal cord, with or without the brainstem; their spatial and temporal scope are subject to technical limitations imposed by low oxygen tension in deep tissues. Therefore, we created an arterially perfused in situ preparation that allowed us to investigate functional interactions in the CNS from the neonatal to adult period. Decerebrated rodents were kept alive via total artificial cardiopulmonary bypass for extracorporeal circulation; the plasma oxygen and ion components needed for survival were supplied through the blood vessels. Interferon regulatory factor 8 (IRF8) is a transcription factor that promotes myeloid cell development and stimulates innate immune responses. In the brain, IRF8 is expressed only in microglia and directs the expression of many genes that serve microglial functions. Recent evidence indicates that IRF8 affects behavior and modulates Alzheimer's disease progression in a mouse model. However, whether this immune deficiency arising from the absence of IRF8 influences the development of the neuronal network in the spinal cord is unknown. We applied the above methodology to mice of all ages and electrophysiologically explored whether the absence of IRF8 influences the development of lumbar central pattern generator (CPG) networks. In mice of all ages, bilateral neuronal discharges by the normal CPG networks activated by the modulated sympathetic tone via descending pathways at high flow rates became organized into discharge episodes punctuated by periods of quiescence. Similar discharge episodes were generated by the adult CPG networks (≥P14 days) activated by drug application. However, discharge episodes elicited by activating the neonatal-juvenile CPG networks (

Transcriptomics and quantitative proteomics reveal changes after second stimulation of bone marrow-derived macrophages from lupus-prone MRL/lpr mice.

Innate immune memory can cause the occurrence and exacerbation of autoimmune diseases, and it is as well as being strongly associated with the pathogenesis of systemic lupus erythematosus (SLE), however, the specific mechanism remains to be further studied. We learned that IFN-γ stimulation generated innate immune memory in bone marrow-derived macrophages (BMDMs) and activated memory interferon-stimulated genes (ISGs). This research used IFN-γ and lipopolysaccharide (LPS) to treat BMDMs with lupus-prone MRL/lpr mice and showed that particular memory ISGs were substantially elevated in prestimulated macrophages. In order to identify the differentially expressed genes (DEGs), researchers turned to RNA-seq. GO and KEGG analysis showed that up-regulated DEGs were enriched in defense and innate immune responses, and were related to the expression of pattern recognition receptors (PRRs)-related pathways in macrophages. TMT-based proteome analysis revealed differentially expressed proteins (DEPs) up-regulated in BMDMs were abundant in metabolic pathways such as glucose metabolism. Our study found that after the secondary stimulation of MRL/lpr mice, the expression of PRRs in innate immune cells was changed, and IFN-related pathways were activated to release a large number of ISGs to promote the secondary response. At the same time, related metabolic modes such as glycolysis were enhanced, and epigenetic changes may occur. Therefore, SLE is brought on, maintained, and worsened by a variety of factors that work together to produce innate immune memory.

Chromatin structure undergoes global and local reorganization during murine dendritic cell development and activation.

Classical dendritic cells (cDCs) are essential for immune responses and differentiate from hematopoietic stem cells via intermediate progenitors, such as monocyte-DC progenitors (MDPs) and common DC progenitors (CDPs). Upon infection, cDCs are activated and rapidly express host defense-related genes, such as those encoding cytokines and chemokines. Chromatin structures, including nuclear compartments and topologically associating domains (TADs), have been implicated in gene regulation. However, the extent and dynamics of their reorganization during cDC development and activation remain unknown. In this study, we comprehensively determined higher-order chromatin structures by Hi-C in DC progenitors and cDC subpopulations. During cDC differentiation, chromatin activation was initially induced at the MDP stage. Subsequently, a shift from inactive to active nuclear compartments occurred at the cDC gene loci in CDPs, which was followed by increased intra-TAD interactions and loop formation. Mechanistically, the transcription factor IRF8, indispensable for cDC differentiation, mediated chromatin activation and changes into the active compartments in DC progenitors, thereby possibly leading to cDC-specific gene induction. Using an infection model, we found that the chromatin structures of host defense-related gene loci were preestablished in unstimulated cDCs, indicating that the formation of higher-order chromatin structures prior to infection may contribute to the rapid responses to pathogens. Overall, these results suggest that chromatin structure reorganization is closely related to the establishment of cDC-specific gene expression and immune functions. This study advances the fundamental understanding of chromatin reorganization in cDC differentiation and activation.

Herbal extracts that induce type I interferons through Toll-like receptor 4 signaling.

BACKGROUND: A mixture of five herbal extracts called internatural (INT), which is prepared from pumpkin seeds, purple turmeric, pearl barley, corn pistil, and cinnamon, is widely used by people in Japan and elsewhere for its immunity-enhancing effects and general health. Although anecdotal evidence indicates its efficacy, the mechanisms by which INT boosts immunity have remained unknown. OBJECTIVE: The aim of this study was to investigate whether INT induces type I interferons (IFNs) in murine bone marrow-derived macrophages (BMDMs) and by what mechanism. DESIGN: We measured induction of type I IFNs (IFNß and IFNα) in BMDMs treated with INT or other Toll-like receptor ligands: bacterial lipopolysaccharides (LPS), dsRNA, poly(I:C), and CpG oligonucleotides. To investigate whether INT signals through Toll-like receptor 4 (TLR4), we tested TLR4-specific inhibitor. We also tested if INT utilizes TLR4 adaptors, toll/IL-1 receptor (TIR) domain-containing adaptor (TRIF), or myeloid differentiation factor 88 (MyD88), we examined INT induction of IFNß in TRIF-KO and MyD88-KO BMDMs. We then investigated whether INT provides an antiviral effect upon fibroblasts either directly or indirectly using the encephalomyocarditis virus (EMCV) model. RESULTS: We first observed that INT, when added to BMDMs, potently induces type I IFNs (IFNß and IFNα) within 2 h. INT induction of IFN expression was mediated by TLR4, which signaled through the TRIF/MyD88 adaptors, similar to LPS. A high-molecular-weight fraction (MW > 10,000) of INT extracts contained IFN-inducing activity. Supernatants from INT-treated BMDMs protected untreated fibroblast from EMCV infection as reduced viral titers. CONCLUSIONS: INT induced type I IFN mRNA and proteins in BMDMs and other cell types. This induction was mediated by TLR4, which transduces signals using the TRIF/MyD88 pathway. The high-MW component of INT contained type I IFN inducing activity. The supernatants from INT-treated cells displayed antiviral activity and protected cells from EMCV infection. These findings indicate that INT is a novel natural IFN inducer that strengthens host's innate immunity.

Bromodomain protein BRD4 directs and sustains CD8 T cell differentiation during infection.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

Innate Immune Memory in Hematopoietic Stem/Progenitor Cells: Myeloid-Biased Differentiation and the Role of Interferon.

Innate immune memory was first described for monocytes and other myeloid cells. This memory is designated Immune Training, in which the host animals that had experienced pathogen infection earlier acquire improved resistance to a second infection. Innate immune memory is mediated by an epigenetic mechanism traced to transcriptional memory that is conserved throughout evolution and has been selected for the ability to mount an adaptive response to shifting environments. Accumulating evidence shows that not only peripheral myeloid cells but hematopoietic stem/progenitor cells (HSCs/HSPCs) can acquire epigenetic memory upon pathogen exposure. Systemic pathogen infection causes HSCs to exit from quiescence and facilitate myeloid-biased differentiation that leads to efficient host defense. This sequence of events is common in HSC memory generation, which is triggered by different stimuli. Recent studies show that not only pathogens but other stimuli such as metabolic stress can generate memory in HSCs. This review summarizes recent publications relevant to HSC memory. We discuss the current understanding of initial sensors, soluble mediators/cytokines involved in memory formation, including Type I and Type II interferons along with future implications.

A RUNX-CBFß-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes.

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX-CBFß-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

Type I IFN-dependent antibody response at the basis of sex dimorphism in the outcome of COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, induces severe pneumonia mainly in elderly males. Epidemiological data clearly indicate sex-based differences in disease outcomes, with men accounting for about 70 % of deaths, despite similar susceptibility to infection. It is well known that females are endowed with higher capacity to produce antibodies, which correlates with viral clearance and disease resolution in the context of SARS-Cov-2 infection. Many X-linked immune genes escape X inactivation showing biallelic expression in female immune cells, particularly in plasmacytoid dendritic cells (pDCs). PDCs are more active in females and endowed with high capability to induce IFN-α-mediated B cell activation and differentiation into antibody-producing plasma cells throughout epigenetic mechanisms linked to trained immunity. Thus, we hypothesize that following SARS-CoV-2 infection, epigenetic modifications of X-linked genes involved in pDC-mediated type I IFN (IFN-I) signaling occurs more effectively in females, for inducing neutralizing antibody response as an immune correlate driving sex-biased disease outcome.

Monocyte Subsets With High Osteoclastogenic Potential and Their Epigenetic Regulation Orchestrated by IRF8.

Osteoclasts (OCs) are bone-resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type (WT) mice, Ly6Chi and Ly6Cint monocytes are the primary source of OC formation when compared to Ly6C- monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8-deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR).

DNA Damage Induces Dynamic Associations of BRD4/P-TEFb With Chromatin and Modulates Gene Transcription in a BRD4-Dependent and -Independent Manner.

The bromodomain-containing protein BRD4 has been thought to transmit epigenetic information across cell divisions by binding to both mitotic chromosomes and interphase chromatin. UV-released BRD4 mediates the recruitment of active P-TEFb to the promoter, which enhances transcriptional elongation. However, the dynamic associations between BRD4 and P-TEFb and BRD4-mediated gene regulation after UV stress are largely unknown. In this study, we found that BRD4 dissociates from chromatin within 30 min after UV treatment and thereafter recruits chromatin. However, P-TEFb binds tightly to chromatin right after UV treatment, suggesting that no interactions occur between BRD4 and P-TEFb within 30 min after UV stress. BRD4 knockdown changes the distribution of P-TEFb among nuclear soluble and chromatin and downregulates the elongation activity of RNA polymerase II. Inhibition of JNK kinase but not other MAP kinases impedes the interactions between BRD4 and P-TEFb. RNA-seq and ChIP assays indicate that BRD4 both positively and negatively regulates gene transcription in cells treated with UV stress. These results reveal previously unrecognized dynamics of BRD4 and P-TEFb after UV stress and regulation of gene transcription by BRD4 acting as either activator or repressor in a context-dependent manner.