Advances in Model Systems for Human Cytomegalovirus Latency and Reactivation

Human cytomegalovirus (HCMV) is a highly prevalent beta-herpesvirus and a significant cause of morbidity and mortality following hematopoietic and solid organ transplant, as well as the leading viral cause of congenital abnormalities. A key feature of the pathogenesis of HCMV is the ability of the virus to establish a latent infection in hematopoietic progenitor and myeloid lineage cells.

Does CD1a Expression Influence T Cell Function in Patients With Langerhans Cell Histiocytosis?

Langerhans cell histiocytosis lesions are characterized by CD1a+ myeloid lineage LCH cells and an inflammatory infiltrate of cytokines and immune cells, including T cells. T cells that recognize CD1a may be implicated in the pathology of many disease states including cancer and autoimmunity but have not been studied in the context of LCH despite the expression of CD1a by LCH cells. In this perspective article, we discuss the expression of CD1a by LCH cells, and we explore the potential for T cells that recognize CD1a to be involved in LCH pathogenesis.

CEBPA Directly Binds Ribosomal DNA and Promotes Ribosomal RNA Transcription in Myeloid Progenitors

Hematopoietic stem cells (HSCs) form a hierarchy of lineage restricted progenitor cells to produce mature hematopoietic cells that vary in function, size, proliferation, and protein synthesis rates. Different hematopoietic cells also vary in the rate of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been identified by their prominent nucleoli, indicating high ribosome biogenesis rates (Fig A). Ribosome biogenesis is an extremely energy intensive process begins with transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA) which further processed into the generation of mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig B). This process is highly dynamic and regulated at the level of rRNA transcription. Despite cell-type and disease-specific variations, rRNA transcription has long been considered a housekeeping process. Hence, cell or tissue type-specific regulation of rRNA transcription has rarely been explored. To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we mapped 2200 publicly available ChIP-Seq datasets representing 249 hematopoietic transcription factors (TFs) and epigenetic factors to create an atlas of hematopoietic TF-rDNA binding. We identified CEBPA that shows consistent and abundant binding to rDNA at a conserved, previously unknown motif in both species (Fig C). CEBPA is a myeloid lineage specific TF whose knockout leads to complete loss of all myeloid lineage cells. It is also frequently mutated (10%) in AML patients. So we picked CEBPA to further characterize its role in rRNA transcription. Since CEBPA deletion causes loss of granulocyte-monocyte progenitors (GMPs), we used the mouse HoxA9-ER cell line (which closely resembles GMPs). To study the immediate consequences of CEBPA loss, We generated a stable degron cell line by biallelically fusing FKBP degron into endogenous loci of Cebpa, enabling to rapidly degrade endogenous CEBPA protein on treatment with dTagV ligand (Fig D, E). To precisely quantify the rate of rRNA transcription, we developed a novel assay called '47S-FISH-Flow' that involves hybridizing fluorescent oligos unique to 5' end of 47S pre-rRNA, which only marks newly synthesized nascent rRNA in the nucleolus, and quantify using flow cytometry (Fig F, G). We found that depleting CEBPA caused rapid decrease in 47S rRNA level and occupancy of Pol I on rDNA (Fig H, I). In summary, we found that myeloid lineage specific TF CEBPA abundantly binds to a conserved motif in rDNA and the depletion of CEBPA rapidly reduces nascent 47S rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Epigenetic Enzyme Mutations in Myeloid Malignancies Are Selected By Chromatin-Remodeling Requirements That Vary By Lineage- and Maturation-Stage

Introduction/Methods: Enzymes that modify histone H3 at lysine 27 (H3K27) to thereby regulate gene transcription (epigenetic enzymes) are recurrently inactivated by deletion and/or mutation in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN) and acute myeloid leukemias (AML). Frustrating understanding of mechanisms is that writers and erasers of the same modification, e.g., methyltransferases that create, and demethylases that remove, H3K27 trimethylation (H3K27me3), a repression ('off') mark, are recurrently inactivated. Moreover, acetyltransferases that write H3K27 acetylation (H3K27ac), an 'on' mark mutually exclusive with H3K27me3, are also recurrently inactivated. One clue to underlying mechanisms is that MDS/MPN/AML present with diverse lineage and maturation phenotypes - perhaps these emerge from, or select for, the diverse epigenetic enzyme mutations. We therefore identified genes upregulated with specific myeloid lineage-commitment, maturation and function fates (~500 genes each) and then examined distributions of H3K27me3 and H3K27ac at these gene-loci in: (i) embryonic stem cells (ESC); (ii) hematopoietic stem and progenitor cells (HSPC); (iii) mature myeloid cells (monocyte [mono], pro-erythroblast, megakaryocyte [MK]); and (iv) AML cells. Results: Terminal-myeloid programs underwent substantial remodeling to gain H3K27ac 'on' mark from ESC/HSPC to mature myeloid (Fig.1A). Providing a mechanism for this, the H3K27 acetyltransferases EP300 and CREBBP were recruited into the RUNX1/SPI1 myeloid-lineage master transcription factor (MTF) hub by cooperation between their transcription activating domains. Mutated/translocated RUNX1, or mutated-NPM1 that cytoplasmically dislocated SPI1, disrupted this cooperation, and reverted hub content to default recruitment of histone deacetylases (HDAC) instead. Demonstrating cause-effect, inhibiting these HDAC renewed AML cell maturation to terminal lineage-fates. Meanwhile, MYC-target (proliferation) genes have high baseline H3K27ac in ESC and HSPC and do not require major remodeling during ontogeny (Fig.1A). An H3K27ac remodeling requirement for lineage-maturation but not proliferation/housekeeping explains selection pressure for inactivating mutations in EP300 or CREBBP, that we found in ~1.2% of MDS/MPN/AML in our (n=690) and other series. Consistent with pan-lineage-maturation needs for H3K27ac, the mutations were found in all lineage sub-types. H3K27me3 'off' mark was mostly erased from myeloid programs in HSPC, but was greater at MK vs erythroid, and also at mono vs granulocyte genes (Fig.1B). This implied more need for H327me3 demethylase (KDM6A/UTX) for HSPC commitment into MK vs erythroid, or mono vs granulocyte, lineages. Accordingly, KDM6A was most upregulated in MK and mono-lineage cells (Fig.1C), and myeloid-conditional Kdm6a knockout decreased platelets and increased red cells in the spleen - reported by others: https://doi.org/10.1182/blood.V128.22.1467.1467. RUNX1-ETO has been shown to specifically impede granulocytic but not mono differentiation - https://www.nature.com/articles/2403396 and KDM6A inactivating mutations were significantly more likely to occur secondary to RUNX1-ETO (4-9% BEAT and AMLSG case series) vs other cytogenetics (0.005%). Selection for KDM6A secondary mutations could thus be to channel myeloid precursors toward lineages most efficiently impeded by primary mutations. Although H3K27me3 was substantially erased at all myeloid-commitment and terminal programs (except MK) in HSPC vs ESC, subsequent lineage-commitment and maturation entailed rewriting H3K27me3 at preceding HSPC and alternate lineage-fate programs, including at MTF genes for alternate fates (Fig.1B, D). Primary MDS/MPN/AML and AML cell lines with inactivating mutations/deletions in H3K27 methyltransferase EZH2 thus displayed aberrant co-expression of lineage MTFs and gene expression programs of normally mutually exclusive lineages (Fig.1E-G). Conclusion. Epigenetic remodeling requirements vary by myeloid lineage and maturation stage. Thus, epigenetic enzyme mutations are selected by, and cause, lineage-context of transformation. This knowledge can guide choice of specific epigenetic enzyme inhibitors to remedy the lineage-maturation defects that drive and define myeloid malignancies. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy; Regeneron: Consultancy; Alexion: Consultancy. Saunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Myeloid Lineage Ablation of Phlpp1 Regulates M-CSF Signaling and Tempers Bone Resorption in Female Mice

Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKOLysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKOLysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that Phlpp1−/− cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates—including Akt, ERK1/2, and PKCζ—accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF.

Distinct Regulations of HO-1 Gene Expression for Stress Response and Substrate Induction

Heme oxygenase-1 (HO-1) is the key enzyme for heme catabolism and cytoprotection. Whereas HO-1 gene expression in response to various stresses has been investigated extensively, the precise mechanisms by which HO-1 gene expression is regulated by the HO-1 substrate heme remain elusive. To systematically examine whether stress-mediated induction and substrate-mediated induction of HO-1 utilize similar or distinct regulatory pathways, we developed an HO-1-DsRed-knock-in reporter mouse in which the HO-1 gene is floxed by loxP sites and the DsRed gene has been inserted. Myeloid lineage-specific recombination of the floxed locus led to fluorescence derived from expression of the HO-1-DsRed fusion protein in peritoneal macrophages. We also challenged general recombination of the locus and generated mice harboring heterozygous recombinant alleles, which enabled us to monitor HO-1-DsRed expression in the whole body in vivo and ex vivo . HO-1 inducers upregulated HO-1-DsRed expression in myeloid lineage cells isolated from the mice. Notably, analyses of peritoneal macrophages from HO-1-DsRed mice lacking NRF2, a major regulator of the oxidative/electrophilic stress response, led us to identify NRF2-dependent stress response-mediated HO-1 induction and NRF2-independent substrate-mediated HO-1 induction. Thus, the HO-1 gene is subjected to at least two distinct levels of regulation, and the available lines of evidence suggest that substrate induction in peritoneal macrophages is independent of CNC family-based regulation.

Lack of Interferon Regulatory Factor 8 Associated with Restricted IFN-gamma expression Augmented Japanese Encephalitis Virus Replication in the Mouse Brain

Interferon Regulatory Factor 8 (IRF8), a myeloid lineage transcription factor, emerges as an essential regulator for microglia activation. However, the precise role of IRF8 during Japanese encephalitis virus (JEV) infection in the brain remains elusive. Here we report that JEV infection enhances IRF8 expression in the infected mice brain. Comparative transcriptional profiling of whole-brain RNA analysis and validation by qRT-PCR reveals an impaired IFNγ and related gene expression in Irf8 knockout ( Irf8 -/- ) infected mice. Further, Ifnγ knockout ( Ifnγ -/- ) mice exhibit a reduced level of Irf8. Both Ifnγ -/- and Irf8 -/- mice exhibit significantly reduced levels of activated (CD11b + CD45 hi , CD11b + CD45 lo , Cd68, and CD86 ) and infiltrating immune cells (Ly6C + , CD4, and CD8) in the infected brain as compared to WT mice. However, a higher level of granulocyte cells (Ly6G + ) infiltration is evident in Irf8 -/- mice and the increased concentration of TNFα, IL6, MCP1 levels in the brain. Interestingly, neither Irf8 -/- nor Ifnγ -/- has conferred protection against lethal JEV challenge to mice and exhibits augmentation in JEV replication in the brain. The gain of function of Irf8 by overexpressing functional IRF8 in an IRF8 deficient cell line attenuates viral replication and enhances IFNγ production. Overall, we summarise that in the murine model of JEV encephalitis, IRF8 modulation affects JEV replication. We also evidence that lack of Irf8 affects immune cells abundance in circulation and the infected brain leading to a reduction in IFNγ level and increased viral load in the brain. Importance Microglial cells, the resident macrophages in the brain, play a vital role in Japanese encephalitis virus (JEV) pathogenesis. The deregulated activity of microglia can be lethal for the brain. Therefore, it is crucial to understand the regulators that drive microglia's phenotype changes and induce inflammation in the brain. Interferon regulatory factor 8 (IRF8) is a myeloid lineage transcription factor involved in microglial activation. However, the impact of IRF8 modulation on JEV replication remains elusive. Moreover, the pathways regulated by IRF8 to initiate and amplify pathological neuroinflammation are not well understood. Here, we demonstrated the effect of IRF8 modulation on JEV replication, microglial activation, and immune cells infiltration in the brain.

Immunoporosis: Role of Innate Immune Cells in Osteoporosis

Osteoporosis or porous bone disorder is the result of an imbalance in an otherwise highly balanced physiological process known as ‘bone remodeling’. The immune system is intricately involved in bone physiology as well as pathologies. Inflammatory diseases are often correlated with osteoporosis. Inflammatory mediators such as reactive oxygen species (ROS), and pro-inflammatory cytokines and chemokines directly or indirectly act on the bone cells and play a role in the pathogenesis of osteoporosis. Recently, Srivastava et al. (Srivastava RK, Dar HY, Mishra PK. Immunoporosis: Immunology of Osteoporosis-Role of T Cells. Frontiers in immunology. 2018;9:657) have coined the term “immunoporosis” to emphasize the role of immune cells in the pathology of osteoporosis. Accumulated pieces of evidence suggest both innate and adaptive immune cells contribute to osteoporosis. However, innate cells are the major effectors of inflammation. They sense various triggers to inflammation such as pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), cellular stress, etc., thus producing pro-inflammatory mediators that play a critical role in the pathogenesis of osteoporosis. In this review, we have discussed the role of the innate immune cells in great detail and divided these cells into different sections in a systemic manner. In the beginning, we talked about cells of the myeloid lineage, including macrophages, monocytes, and dendritic cells. This group of cells explicitly influences the skeletal system by the action of production of pro-inflammatory cytokines and can transdifferentiate into osteoclast. Other cells of the myeloid lineage, such as neutrophils, eosinophils, and mast cells, largely impact osteoporosis via the production of pro-inflammatory cytokines. Further, we talked about the cells of the lymphoid lineage, including natural killer cells and innate lymphoid cells, which share innate-like properties and play a role in osteoporosis. In addition to various innate immune cells, we also discussed the impact of classical pro-inflammatory cytokines on osteoporosis. We also highlighted the studies regarding the impact of physiological and metabolic changes in the body, which results in chronic inflammatory conditions such as ageing, ultimately triggering osteoporosis.