Transcription Factor PU Research Articles

FBW7 Inhibits Myeloid Differentiation in Acute Myeloid Leukemia via GSK3-Dependent Ubiquitination of PU.1.

Glycogen synthase kinase 3β (GSK3β), an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including acute myeloid leukemia (AML) where it promotes self-renewal, growth, and survival of AML cells. Therefore, GSK3β inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte-macrophage differentiation pathway as potential GSK3β target. We demonstrate that GSK3β phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte-macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41, S140A retained its transactivation, DNA-binding ability and promoted monocyte-macrophage differentiation of U937 cells even without phorbol 12-myristate 13-acetate (PMA) treatment. We further showed that FBW7 overexpression inhibited both PMA as well as M-CSF-induced macrophage differentiation of myeloid cell lines and peripheral blood mononuclear cells (PBMC) from healthy volunteers, respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3β-FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from patients with leukemia where FBW7 overexpression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared with M-CSF treatment alone. IMPLICATIONS: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3β inhibition and further substantiates potential of GSK3β as a therapeutic target in AML.

PHF6 Restricts AML Acceleration By Promoting Myeloid Differentiation Genes in Leukemic Cells

Acute myeloid leukemia is caused by the accumulation of mutations in hematopoietic stem and myeloid progenitor cells, resulting in increased self-renewal, inhibition of differentiation, and aberrant proliferation. Although genomic studies have comprehensively identified genes that are mutated in acute leukemias, the functional roles of many of them, and the consequences of their mutations, remain poorly understood. PHF6 (PHD-finger protein 6) is an X-linked gene that is mutated in 3.2% of de novo AML, 4.7% CMML, 3% MDS, and 1.6% CML patients. Two-thirds of somatic mutations in PHF6 are frameshift and nonsense mutations distributed throughout the gene body, resulting in loss of PHF6 protein. One-third of the mutations are point mutations clustered in the ePHD2 (extended PHD) domain, and the consequence of these mutations on PHF6 function is unknown. The functional role of PHF6 and the mechanism by which PHF6 mutations accelerate AML has not yet been determined. In this study, we delineate the cellular and molecular function of PHF6 in AML using in vitro and in vivo models. In agreement with recently published reports, we found that pan-hematopoietic deletion of Phf6 using the Vav-Cre recombinase system gave competitive transplantation advantage to HSCs, with sustained multi-lineage reconstitution without exhaustion over three rounds of serial transplantations, demonstrating that Phf6 represses HSC self-renewal. However, loss of Phf6 alone was insufficient to cause hematopoietic malignancy in the mouse model when monitored for one year. To determine the function of PHF6 in AML progression, we transduced cKO (Vav-Cre; Phf6 flox) or WT (Vav-Cre only) bone marrow cells with MSCV retrovirus expressing HOXA9 (WT+HOXA9 and cKO+HOXA9), and transplanted into irradiated recipient mice. The resulting HOXA9-driven AML was greatly accelerated in the Phf6 cKO background, with recipient mice succumbing faster (median survival 119 days) as compared to recipients transduced with HOXA9-transduced WT cells (median survival >180 days, p=0.003) (Fig 1A). This was also reflected by an increase in the number of circulating immature leukemic cells in peripheral blood at earlier timepoints. HOXA9-transduced cKO cells showed higher serial replating ability in an in vitro colony forming assay as compared with HOXA9-transduced WT cells (Fig 1B). We further investigated the molecular function of PHF6 using the THP-1 human AML cell line. PHF6 is a chromatin-binding protein with two ePHD domains, and its binding partners and pattern of chromatin occupancy are unclear. Using ChIP-Seq, we identified that PHF6 occupies enhancers, and its peaks show striking alignment with the peaks of the key myeloid transcription factors (TFs) RUNX1, PU.1, and IRF8 (Fig 1C). To assess the effect of the clinically relevant point mutation R274Q (in the ePHD2 domain) on the transcriptional effects produced by PHF6, we first generated a PHF6 KO clone from the THP-1 cell line, and then re-expressed either WT PHF6 or R274Q-mutant PHF6 in this KO line. Re-expression of WT PHF6 rescued the extensive gene expression changes produced by its knockout, but R274Q-mutant PHF6 was unable to produce any gene expression changes, indicating that it is a "transcriptionally dead" mutant (Fig 1D). Gene Ontology analysis of transcriptome changes induced by WT PHF6 showed that PHF6 promotes the expression of myeloid differentiation gene sets. In summary, PHF6 restricts AML progression by binding enhancers with key myeloid TFs, and promoting the expression of myeloid differentiation genes. R274Q mutation renders PHF6 unable to exert any downstream expression changes, indicating that the ePHD2 domain (where R274 is located, clustered with other amino acids showing point mutations in hematopoietic malignancies) is critical for PHF6 function, and likely mediates important functional interactions with chromatin partners. Our future work will involve dissection of the sequence of molecular events governed by PHF6 following enhancer occupancy, and the role of PHF6 in repressing AML self-renewal and promoting differentiation. Disclosures No relevant conflicts of interest to declare.

Core Binding Factor Leukemias Utilize a Physiologic Sense/Antisense Promoter Switch Employed By T-Cells

Introduction: Alterations of core binding factors (CBF), Runx1 and CBFβ are frequent mutational targets in acute myeloid leukemia (AML). Chromosomal translocations t(8;21)(q22;q22) and inv(16)(p13q22), creating the fusion proteins RUNX1-ETO and CBFβ-MYH11 respectively, account for 15% and thus the largest sub-group of AML called CBF-AML. CBF oncogenes induce global changes in chromatin structure and gene regulation, which lead to differentiation blockade. A critical leukemic event could be the inactivation of PU.1 transcription factor. Normal myeloid differentiation needs PU.1 levels to increase, failure to do so leads to a stop of differentiation and AML development. In contrast, T-cell differentiation requires PU.1 to be completely switched off. The exact mechanism of PU.1 suppression, physiological for T-lymphopoiesis or pathological for leukemia, remains elusive. Results: We assessed the activation of the PU.1 locus throughout human hematopoietic differentiation stages using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Interestingly, we observed high accessibility of a previously identified antisense promoter (AsPr) in intron 3 and antisense transcript (asRNA) expression during early lymphopoiesis which preceded locus shutdown in T cells. The ratio of AsPr/PrPr accessibility and of antisense/sense transcription clearly indicated cellular fate during hematopoiesis (Figure 1A). T-lymphoid differentiation was related to the timely expression of RUNX transcription factors. RUNX1, RUNX3, and the CBF fusions RUNX1-ETO and CBFβ-MYH11 were capable to transactivate PU.1 AsPr. In CBF-AML patient samples we strikingly found elevated asRNA/mRNA ratios compared to normal karyotype AML or healthy CD34+ cells (Figure 1B) and increased AsPr/PrPr ratios unsing DNaseI-seq data in RUNX1-ETO AML patients. Functionally we found that PU.1 asRNA depletion in t(8;21) xenografted immune-deficient (NOD/SCID) mice restored a normal survival (Figure 1C) demonstrating that PU.1 antisense transcripts are required for CBF leukemia outgrow in vivo. To further dissect the mechanism of how CBFs could drive PU.1 antisense transcription we applied active RNA polymerase mapping (PRO-seq) and chromatin accessibility (ATAC-seq) and found a shift from PU.1 antisense to sense transcription after RUNX1-ETO depletion. Using chromosomal conformation capture sequencing (3C, Hi-C and CHiC) in T-lymphoid, myeloid and RUNX1-ETO cells combined with transcript quantification we observed that competitive interaction of an upstream enhancer with the proximal or the antisense promoter are at the heart of differential PU.1 expression during myeloid and T-cell development (Figure 2A). Leukemic CBF fusions thus utilize a physiologic mechanism employed by T-cells to decrease sense PU.1 transcription (Figure 2B). Conclusion: The data suggest that silencing transcription factor PU.1 is an active process that requires a specific chromosome formation that is induced by CBF fusions. Sense/antisense promoter competition represents a crucial functional switch for gene expression perturbation by oncogenes and provide a potential strategy for future precise therapeutic targeting of oncogene-induced chromatin remodeling. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Staber:Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; msd: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria.

Transcription factor PU.1 and immune cell differentiation (Review).

The transcription factor PU.1, an important member of the ETS family, plays a significant role in the differentiation of immune cells, which include macrophages, neutrophils, dendritic cells, T lymphoid cells, B lymphoid cells and so on. Immune cells are involved in the occurrence and development of diseases, including inflammatory diseases, neoplastic diseases and immune diseases. Therefore, it is particularly crucial to elucidate the roles and mechanisms of PU.1 in immune cells. The elucidation of these mechanisms may lead to the development of more effective therapeutic strategies for the treatment of inflammatory diseases and immune‑mediated diseases mediated by various immune cells. With the development of molecular biology, the mechanisms of PU.1 in immune cell differentiation have been further explained. Different levels of PU.1 expression determine the type of immune cell differentiation. PU.1 expression is increased during granulocyte and macrophage differentiation, while it is decreased during T lymphocyte and B lymphocyte differentiation. The present study reviews and discusses the effects of the transcription factor PU.1 on immune cell differentiation.

Pterostilbene reduces colonic inflammation by suppressing dendritic cell activation and promoting regulatory T cell development.

Dendritic cells (DCs) and T cells play important roles in immune regulation, and modulating their function is an approach for developing preventive or therapeutic strategies against immune disorders. Herein, the effect of pterostilbene (PSB) (3',5'-dimethoxy-resveratrol)-a resveratrol-related polyphenol found in blueberries-on immune regulation was evaluated. Using an in vitro co-culture system, PSB was found to exert the strongest inhibitory effect among all tested resveratrol derivatives on DC-mediated T cell proliferation; moreover, PSB treatment decreased the Th1 and Th17 populations and increased the regulatory T cell (Treg) population. Upon co-stimulation with anti-CD3 and anti-CD28 antibodies, PSB inhibited CD4+ T cell proliferation and differentiation into Th1 cells. Additionally, PSB acted on DCs to suppress the lipopolysaccharide-induced transactivation of genes encoding antigen presentation-related molecules and inflammatory cytokines by attenuating the DNA-binding ability of the transcription factor PU.1. Furthermore, PSB promoted DC-mediated Foxp3+ Treg differentiation, and PU.1 knockdown increased DC-induced Treg activity. Oral administration of PSB alleviated the symptoms of dextran sulfate sodium-induced colitis and decreased tumor necrosis factor-α expression in mice. Thus, PSB treatment ameliorates colonic inflammation.

Abstract P140: Myeloid Activation in Hypertension and Effects on Bone: A New End Organ?

Hypertension affects approximately one-half of the adult population. Another common disease is osteoporosis, which affects 53 million people in the United States. Clinical studies have shown an association between hypertension and bone loss. We hypothesized that experimental hypertension would be associated with osteoporosis. In Angiotensin (Ang) II-induced hypertension, there is an increase in hematopoietic stem cells (HSCs) expressing the master myeloid transcription factor PU.1 (2.99 ± 0.92) vs. sham (0.17 ± 0.04,p=0.007). Here we reported an increase an osteoclasts number per mm 2 by Tartrate-resistant acid phosphatase staining in DOCA-salt hypertension (27.4 ± 2.3) vs. sham (12.09 ± 1.7, p=0.0002). Using microCT analysis of femoral bone, we found that Ang II-induced hypertension caused striking bone loss as reflected by decreases in cortical bone area, cortical thickness and trabecular number (see Table). We also examined bone strength using a three-point biomechanical testing. In keeping with the micro-CT data, both forms of hypertension were associated with increased bone fragility, reflected by several parameters including maximum force to failure and rigidity (see Table). Recent studies have implicated T cells in the genesis of osteoporosis, and in keeping with this, we found almost two-fold increase in both CD4 + and CD8 + T cells in the marrow of mice with DOCA-salt hypertension. Thus, in two models of experimental hypertension there is marked bone loss and the development of bone fragility, potentially related to T cell accumulation and cytokine release. These findings might explain the association between osteoporosis and hypertension.

Alternative Activation of Macrophages Is Accompanied by Chromatin Remodeling Associated with Lineage-Dependent DNA Shape Features Flanking PU.1 Motifs.

IL-4 activates macrophages to adopt distinct phenotypes associated with clearance of helminth infections and tissue repair, but the phenotype depends on the cellular lineage of these macrophages. The molecular basis of chromatin remodeling in response to IL-4 stimulation in tissue-resident and monocyte-derived macrophages is not understood. In this study, we find that IL-4 activation of different lineages of peritoneal macrophages in mice is accompanied by lineage-specific chromatin remodeling in regions enriched with binding motifs of the pioneer transcription factor PU.1. PU.1 motif is similarly associated with both tissue-resident and monocyte-derived IL-4-induced accessible regions but has different lineage-specific DNA shape features and predicted cofactors. Mutation studies based on natural genetic variation between C57BL/6 and BALB/c mouse strains indicate that accessibility of these IL-4-induced regions can be regulated through differences in DNA shape without direct disruption of PU.1 motifs. We propose a model whereby DNA shape features of stimulation-dependent genomic elements contribute to differences in the accessible chromatin landscape of alternatively activated macrophages on different genetic backgrounds that may contribute to phenotypic variations in immune responses.

High Amount of Transcription Factor IRF8 Engages AP1-IRF Composite Elements in Enhancers to Direct Type 1 Conventional Dendritic Cell Identity

Development and function of conventional dendritic cell (cDC) subsets, cDC1 and cDC2, depend on transcription factors (TFs) IRF8 and IRF4, respectively. Since IRF8 and IRF4 can each interact with TF BATF3 at AP1-IRF composite elements (AICEs) and with TF PU.1 at Ets-IRF composite elements (EICEs), it is unclear how these factors exert divergent actions. Here, we determined the basis for distinct effects of IRF8 and IRF4 in cDC development. Genes expressed commonly by cDC1 and cDC2 used EICE-dependent enhancers that were redundantly activated by low amounts of either IRF4 or IRF8. By contrast, cDC1-specific genes relied on AICE-dependent enhancers, which required high IRF concentrations, but were activated by either IRF4 or IRF8. IRF8 was specifically required only by a minority of cDC1-specific genes, such as Xcr1, which could distinguish between IRF8 and IRF4 DNA-binding domains. Thus, these results explain how BATF3-dependent Irf8 autoactivation underlies emergence of the cDC1-specific transcriptional program.

Notch2 complements Notch1 to mediate inductive signaling that initiates early T cell development.

Notch signaling is the dominant intercellular signaling input during the earliest stages of T cell development in the thymus. Although Notch1 is known to be indispensable, we show that it does not mediate all Notch signaling in precommitment stages: Notch2 initially works in parallel to promote early murine T cell development and antagonize other fates. Notch-regulated target genes before and after T lineage commitment change dynamically, and we show that this partially reflects shifts in genome-wide DNA binding by RBPJ, the transcription factor activated by complex formation with the Notch intracellular domain. Although Notch signaling and transcription factor PU.1 can activate some common targets in precommitment T progenitors, Notch signaling and PU.1 activity have functionally antagonistic effects on multiple targets, delineating separation of pro-T cells from alternative PU.1-dependent fates. These results define a distinct mechanism of Notch signal response that distinguishes the initial stages of murine T cell development.

Dual Roles of PU.1 in the Expression of PD-L2: Direct Transactivation with IRF4 and Indirect Epigenetic Regulation

PD-L2, which has been identified as a PD-1 ligand, is specifically expressed in dendritic cells (DCs) and macrophages. The transcription factors that determine the cell type-specific expression of PD-L2 are largely unknown, although PD-1 and its ligands, which have been shown to play important roles in T cell suppression, have been vigorously analyzed in the field of cancer immunology. To reveal the mechanism by which Pdcd1lg2 gene expression is regulated, we focused on DCs, which play key roles in innate and acquired immunity. The knockdown of the hematopoietic cell-specific transcription factors PU.1 and IRF4 decreased PD-L2 expression in GM-CSF-induced mouse bone marrow-derived DCs. Chromatin immunoprecipitation assays, luciferase assays, and electrophoretic mobility shift assays demonstrated that PU.1 and IRF4 bound directly to the Pdcd1lg2 gene via an Ets-IRF composite element sequence and coordinately transactivated the Pdcd1lg2 gene. Furthermore, PU.1 knockdown reduced the histone acetylation of the Pdcd1lg2 gene. The knockdown of the typical histone acetyltransferase p300, which has been reported to interact with PU.1, decreased the expression and H3K27 acetylation of the Pdcd1lg2 gene. GM-CSF stimulation upregulated the Pdcd1lg2 gene expression, which was accompanied by an increase in PU.1 binding and histone acetylation in Flt3L-generated mouse bone marrow-derived DCs. The involvement of PU.1, IRF4, and p300 were also observed in mouse splenic DCs. Overall, these results indicate that PU.1 positively regulates Pdcd1lg2 gene expression as a transactivator and an epigenetic regulator in DCs.

3069 – GRANULIN, REVEALED BY SINGLE-CELL RNA-SEQUENCING OF DEVELOPING HEMATOPOIETIC CELLS, DRIVES MACROPHAGE AND NEUTROPHIL DIFFERENTIATION

Myeloid cell differentiation leads to the formation of neutrophils and macrophages, which are critical cells for host defenses against infections. In addition, a multitude of other functions beyond their classical inflammatory roles have been assigned to these cells, including wound healing, neurodegenerative diseases and tumorigenesis. Thus, the identification of novel genes that regulate myeloid cell development has undoubtedly the potential to impact diverse areas of human health. Here, we have used deep single-cell RNA-sequencing during the active phase of myelopoiesis in the zebrafish embryo to create a platform for the identification of novel genes required for myeloid cell formation. Using this platform, we have uncovered a role for granulin (GRN) during neutrophil and macrophage differentiation. Leveraging the restricted expression of the zebrafish orthologue grna to the myeloid cell lineage, enabled us to perform loss of function experiments without altering its function in non-hematopoietic cells. With the generation of a zebrafish model of grna deficiency, we reveal that myeloid progenitors are unable to differentiate into neutrophils and macrophages. These grna deficient progenitors are also incapable of triggering a myelopoiesis emergency response, resulting in abnormal wound healing with aberrant collagen depositions. Utilizing CUT&RUN, we identified that Pu.1 directly binds grna enhancers, triggering its expression. Similar to the zebrafish grna, the mammalian granulin is also upregulated in myeloid cells, and controlled by the myeloid transcription factors PU.1 and IRF8. Altogether, our findings uncover the role of Granulin during myeloid cell differentiation, opening a new field of study that will help elucidate the pleiotropic role of this enigmatic protein in inflammation, wound healing, tumor progression and neurodegenerative disease.

Mechanistic action of the acute toxicity of Bajitian (Morinda officinalis) in zebrafish embryos

Abstract Objectives To investigate acute toxicity of Bajitian (Morinda officinalis) in zebrafish embryos. Methods Zebrafish embryos at 48-h post fertilization (hpf) were exposed to Bajitian ethanol extract for 72 h. The causative action of a delay in yolk sac absorption by Bajitian was investigated by RT-PCR analysis of lipid metabolism-related microsomal triglyceride transfer protein (MTP), apolipoprotein C-II (ApoC2) and lipogenesis-related liver x receptor (LXR) genes. The effect of Bajitian eliciting an inflammatory response was studied by exposing 72 hpf myeloperoxidase (MPO): GFP transgenic zebrafish embryos to Bajitian extract for 4 h. Assessment was done by TUNEL, caspase-3/7, and RT-PCR analysis of the apoptosis related pathway B-cell lymphoma 2 associated X protein (Bax), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) genes, neutrophil development-related stem cell leukaemia (SCL) and transcription factor PU.1 genes, to reveal the causative action of Bajitian reducing neutrophils. Results RT-PCR analysis found that Bajitian extract had no effect on the expression of MTP or ApoC2 genes, but upregulated LXR gene, which might explain the delay in yolk sac absorption. Analysis of the inflammatory response showed that compared with negative controls, Bajitian extract significantly (P Conclusions This study demonstrated that Bajitian caused a delay in yolk sac absorption and a decrease neutrophil in zebrafish embryos, which may be related to the inhibition of neutrophil development.

Myelopoiesis specific gene expression profiling in human CD34+ hematopoietic stem cells

Differentiation of the HSCs into myeloid lineage is primarily monitored by transcription factor PU.1. GATA1 acts as a negative regulator by antagonizing the function of PU.1 by bindings its β3/β4 domain. In this study, a mutation was induced in PU.1 which blocks its interaction with GATA1. The pure form of this mutant protein i.e Y244D was loaded on poly (lactic-co-glycolic acid) nanoparticles to transfect CD34+ cells, which act as a selective tool to enhance the myelopoiesis, as confirmed by flow cytometry analysis. Further, microarray data analysis was performed to gather information on myelopoiesis specific signaling pathways and genes involved in myelopoiesis like CCL2, S100A8, and S100A9, which were also found to be significantly upregulated in the mutant form. Different molecular functions like antioxidant activity, signal transduction, developmental processes, and biological adhesion were analyzed. This study potentially signifies that PU.1 mutant is highly efficient in myelopoiesis.

Niclosamide and its derivative DK-520 inhibit RANKL-induced osteoclastogenesis.

Niclosamide is a potent inhibitor of osteoclastogenesis and bone remodeling. DK-520 is an acyl derivative of Niclosamide and significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. However, at present the effect of DK-520 on osteoclastogenesis has not been reported. Here, we investigated whether DK-520 can regulate receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis of bone marrow macrophages (BMMs) invitro. Following induction of BMMs with RANKL for three days, we detected differentiated osteoclasts with typical morphology and high levels of tartrate-resistant acid phosphatase (TRAP), RANKL, and cathepsin K (CTSK) expression. Treatment with either Niclosamide or DK-520 did not affect the viability of osteoclast precursors (OCPs), but significantly inhibited RANKL-induced transdifferentiation of macrophages into OCPs, particularly in the early stage of osteoclastogenesis. Both Niclosamide and DK-520 significantly decreased the relative levels of transcription factor PU.1 mRNA transcripts and dendritic cell-specific transmembrane protein (DC-STAMP), but not v-ATPasev0 d2 protein expression in OCPs. In addition, the inhibitory effect of DK-520 on osteoclastogenesis is realized through impairment of the NF-kB (nuclear factor-κB) and MAPK (mitogen-activated protein kinase) signaling pathways. These results demonstrate that DK-520, like Niclosamide, effectively inhibits the early stage of osteoclastogenesis. The findings presented here, together with its increased oral plasma concentrations and bioavailability, suggest that DK-520 may be a promising drug candidate for treatment of osteoclast-related diseases.

From the Editor's Desk…

From the Editor's Desk…

Targeting Microglial Population Dynamics in Alzheimer's Disease: Are We Ready for a Potential Impact on Immune Function?

Alzheimer’s disease (AD) is the most common form of dementia, affecting two-thirds of people with dementia in the world. To date, no disease-modifying treatments are available to stop or delay the progression of AD. This chronic neurodegenerative disease is dominated by a strong innate immune response, whereby microglia plays a central role as the main resident macrophage of the brain. Recent genome-wide association studies (GWASs) have identified single-nucleotide polymorphisms (SNPs) located in microglial genes and associated with a delayed onset of AD, highlighting the important role of these cells on the onset and/or progression of the disease. These findings have increased the interest in targeting microglia-associated neuroinflammation as a potentially disease-modifying therapeutic approach for AD. In this review we provide an overview on the contribution of microglia to the pathophysiology of AD, focusing on the main regulatory pathways controlling microglial population dynamics during the neuroinflammatory response, such as the colony-stimulating factor 1 receptor (CSF1R), its ligands (the colony-stimulating factor 1 and interleukin 34) and the transcription factor PU.1. We also discuss the current therapeutic strategies targeting proliferation to modulate microglia-associated neuroinflammation and their potential impact on peripheral immune cell populations in the short and long-term. Understanding the effects of immunomodulatory approaches on microglia and other immune cell types might be critical for developing specific, effective, and safe therapies for neurodegenerative diseases.

Th9 Cells in Peripheral Blood Increased in Patients with Immune-Related Pancytopenia.

Background Immune-related pancytopenia (IRP) is a kind of autoimmune disease mediated by autoantibodies in bone marrow. T helper 9 (Th9) cell is a new subset of T cell discovered recently, which mainly expresses cytokine interleukin-9 (IL-9) to exert immune function. Th9 cells are associated with a variety of inflammatory diseases, but the role of Th9 cells in IRP remains unclear. Methods Fifty patients with IRP and 20 healthy controls were enrolled. The percentage of Th9 cells was detected by flow cytometry (FCM) and ELISA. CD4+ lymphocytes were sorted by magnetic beads, and the mRNA expression levels of Th9 cells related transcription factors PU.1 and BATF were detected by RT-PCR. Results The percentage of Th9 cells in CD3+CD4+ cells was 2.73 ± 1.96% in the untreated group, which was significantly higher than those in the remission group (1.21 ± 0.86%) (p < 0.01) and the control group (0.68 ± 0.40%) (p < 0.001). And that in the remission group was significantly higher than that in the control group (p < 0.05). The level of IL-9 in the untreated group was 183.91 ± 112.42 pg/mL, which was significantly higher than that in the remission group (105.96 ± 64.79 pg/mL) (p < 0.01) and control group (56.03 ± 14.49 pg/mL) (p < 0.001). That in the remission group was also significantly higher than that in the control group (p < 0.01). They were negatively correlated with hemoglobin, red blood cell, white blood cell, and platelet counts and positively correlated with the percentage of CD19+B cells and CD5+CD19+/CD19+B cells, respectively. The mRNA expression levels of PU.1 and BATF in IRP patients were higher than those in controls (p < 0.05). Conclusions The percentage of Th9 cells in the peripheral blood and the level of IL-9 in the serum of patients with IRP were increased, which was related to the severity of the disease.

PU.1 mediates hematopoietic stem cell self-renewal during cellular stress by non-canonical Wnt signaling

Abstract Hematopoietic stem cells (HSCs) reside in a dormant state during tissue homeostasis. However, secreted niche factors and cellular programs that maintain HSC dormancy following hematopoietic stress remain unknown. HSC are capable of re-entering quiescence following proliferation, suggesting local factors control HSC exhaustion. In the present study, we found that the myeloid-associated transcription factor PU.1 alters cell cycle activity and differentiation during cellular stress. PU.1 is typically associated with myeloid differentiation and lineage commitment, but during HSC perturbation, PU.1 regulates HSC maintenance and self-renewal potential. Taken together, PU.1 is required to limit HSC exhaustion; furthermore, limiting PU.1 expression during stress leads to impaired HSC function. Using cell-specific reporter models and reductionist culture assays, we find that a non-canonical Wnt-PU.1 axis constrains HSC differentiation and activation to maintain tissue homeostasis. Moreover, using scRNAseq and ChIPseq, we found unique cellular programs associated with PU.1 localization that mediate cell cycling and metabolism of proliferative HSC are mediated by Wnt. In the context of cellular stress, exposure to non-canonical Wnt signaling transitions HSCs into PU.1 expressing cells with increased engraftment potential and multilineage reconstitution. Our results suggest Wnt-induced PU.1 expression limit proliferation and differentiation programs to promote HSC function. Altogether, our data identify PU.1 as a key mediator of HSC maintenance in response to cellular stress.

PU.1 regulates Ccr7 gene expression by binding to its promoter in naïve CD4+ T cells.

C‐C chemokine receptor type 7 (CCR7) is expressed on naïve T cells, B cells, and activated dendritic cells (DCs). We previously demonstrated that the transcription factor PU.1/Spi1 positively regulates the expression of CCR7 in DCs. In the present study, we investigated the role of PU.1 in CCR7 expression in T cells. To confirm whether PU.1 is involved in the expression of CCR7, we conducted a ChIP assay in various T cells purified from splenocytes and thymocytes and found that PU.1 binds to the Ccr7 promoter‐proximal region in spleen naïve CD4+ T cells, but not in thymocytes. Small interfering RNA‐mediated PU.1 knockdown resulted in decreased CCR7 expression in spleen naïve CD4+ T cells. Compared to naïve CD4+ T cells, Spi1 and Ccr7 mRNA levels decreased in Th1 and Th2 cells, in which PU.1 did not bind to the Ccr7 promoter, suggesting that CCR7 expression decreases due to the dissociation of PU.1 from the Ccr7 promoter during the development of effector T cells from naïve T cells. Collectively, we concluded that CCR7 expression level correlates with the binding level of PU.1 to the Ccr7 promoter and PU.1 acts as a transcriptional activator of the Ccr7 gene in naïve CD4+ T cells.

Abnormal expression of colony stimulating factor 1 receptor (CSF1R) and transcription factor PU.1 (SPI1) in the spleen from patients with major psychiatric disorders: A role of brain–spleen axis

BackgroundThe colony stimulating factor 1 receptor (CSF1R) regulates microglia/macrophage proliferation, differentiation and survival; however, the precise role of this protein in psychiatric disorders is unknown. CSF1R is also known to interact with the transcription factor PU.1 (SPI1). Here we studied whether the expressions of CSF1R and SPI1 are altered in the postmortem samples (parietal cortex, cerebellum, spleen) from patients with major psychiatric disorders. MethodsProtein expression of CSF1R and SPI1 in the parietal cortex, cerebellum and spleen from control, major depressive disorder (MDD), schizophrenia (SZ), and bipolar disorder (BD) groups was measured. ResultsLevels of CSF1R in the spleen, but not cerebellum and parietal cortex, from MDD and SZ groups were significantly lower than the control group. There was a positive correlation between CSF1R levels in the spleen and CSF1R levels in the parietal cortex in the all subjects from four groups. Furthermore, levels of SPI1 in the cerebellum and spleen from MDD and SZ groups were significantly higher than the control group. Levels of SPI1 in the parietal cortex were not different among the four groups. Interestingly, there was a negative correlation between CSF1R and SPI1 levels in the spleen of the all subjects from four groups. There was also a negative correlation between CSF1R and SPI1 levels in the spleen of MDD group. LimitationsThe small number in each group may limit our interpretation. ConclusionsAbnormalities in CSF1R and SPI1 in the brain–spleen axis might, in part, play a role in the pathophysiology of MDD.