Differentiation Of Monocytic Cells Research Articles

Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody.

CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147KO THP-1 cells and evaluated their differentiation properties compared to the wild type. A humanized anti-CD147 antibody, HuM6-1B9, was also constructed for investing the phagocytic function of CD147KO THP-1 cells mediated by HuM6-1B9 in the phagocytosis of Jurkat T cells. The CD147KO THP-1 was generated by CRISPR/Cas9 and maintained polarization profiles. HuM6-1B9 was produced in CHO-K1 cells and effectively bound to CD147 with high binding affinity (KD: 2.05 ± 0.30 × 10-9 M). Additionally, HuM6-1B9 enhanced the phagocytosis of Jurkat T cells by CD147KO THP-1-derived LPS-activated macrophages (M-LPS), without self-ADCP. The formation of THP-1-derived mMDSC was limited in CD147KO THP-1 cells, highlighting the significant impact of CD147 deletion. Maintaining expression markers and phagocytic function in CD147KO THP-1 macrophages supports future engineering and the application of induced pluripotent stem cell-derived macrophages. The combination of HuM6-1B9 and CD147KO monocyte-derived macrophages holds promise as an alternative strategy for T-ALL.

Targeting SAT1 prevents osteoporosis through promoting osteoclast apoptosis

Osteoporosis is a systemic bone disease characterized by decreased bone mass that is tightly regulated by the coordinated actions of osteoclasts and osteoblasts. Apoptosis as a precise programmed cell death involves a cascade of gene expression events which are mechanistically linked to the regulation of bone metabolism. Nevertheless, the critical biomolecules involved in regulating cell apoptosis in osteoporosis remain unknown. To gain a deeper insight into the relationship between apoptosis and osteoporosis, this study integrated the sequencing results of human samples and using a machine learning workflow to overcome the limitations of a single study. Among all immune cell populations, we assessed the apoptotic level and portrayed the distinct subtypes and lineage differentiation of monocytic cells in osteoporotic tissues. Osteoclasts expressed a higher level of Spermidine/spermine-N1-Acetyltransferase1 (SAT1) during osteoclastogenesis which prevented osteoclasts apoptosis and facilitate osteoporosis progression. In addition, Berenil, one potent SAT1 inhibitor, increased osteoclast apoptosis and reversed the bone loss in the femurs of a murine ovariectomy model. In summary, Berenil promotes osteoclast apoptosis, inhibits the bone resorption and improves the abnormal bone structure in vitro and in vivo models by targeting SAT1, demonstrating its potential as a precise therapeutic strategy for clinical osteoporosis treatment.

Monocytic Differentiation of Human Acute Myeloid Leukemia Cells: A Proteomic and Phosphoproteomic Comparison of FAB-M4/M5 Patients with and without Nucleophosmin 1 Mutations.

Even though morphological signs of differentiation have a minimal impact on survival after intensive cytotoxic therapy for acute myeloid leukemia (AML), monocytic AML cell differentiation (i.e., classified as French/American/British (FAB) subtypes M4/M5) is associated with a different responsiveness both to Bcl-2 inhibition (decreased responsiveness) and possibly also bromodomain inhibition (increased responsiveness). FAB-M4/M5 patients are heterogeneous with regard to genetic abnormalities, even though monocytic differentiation is common for patients with Nucleophosmin 1 (NPM1) insertions/mutations; to further study the heterogeneity of FAB-M4/M5 patients we did a proteomic and phosphoproteomic comparison of FAB-M4/M5 patients with (n = 13) and without (n = 12) NPM1 mutations. The proteomic profile of NPM1-mutated FAB-M4/M5 patients was characterized by increased levels of proteins involved in the regulation of endocytosis/vesicle trafficking/organellar communication. In contrast, AML cells without NPM1 mutations were characterized by increased levels of several proteins involved in the regulation of cytoplasmic translation, including a large number of ribosomal proteins. The phosphoproteomic differences between the two groups were less extensive but reflected similar differences. To conclude, even though FAB classification/monocytic differentiation are associated with differences in responsiveness to new targeted therapies (e.g., Bcl-2 inhibition), our results shows that FAB-M4/M5 patients are heterogeneous with regard to important biological characteristics of the leukemic cells.

The common H232 STING allele shows impaired activities in DNA sensing, susceptibility to viral infection, and in monocyte cell function, while the HAQ variant possesses wild-type properties

Different innate immune pathways converge to Stimulator of interferon genes (STING) and trigger type I interferon responses after recognition of abnormal nucleic acids in the cells. This non-redundant function renders STING a major player in immunosurveillance, and an emerging target for cancer and infectious diseases therapeutics. Beyond somatic mutations that often occur in cancer, the human gene encoding STING protein, TMEM173 (STING1), holds great genetic heterogeneity; R232, HAQ (R71H-G230A-R293Q) and H232 are the most common alleles. Although some of these alleles are likely to be hypomorphic, their function is still debated, due to the available functional assessments, which have been performed in biased biological systems. Here, by using genetic background-matched models, we report on the functional evaluation of R232, HAQ and H232 variants on STING function, and on how these genotypes affect the susceptibility to clinically relevant viruses, thus supporting a potential contributing cause to differences in inter-individual responses to infections. Our findings also demonstrate a novel toll-like receptor-independent role of STING in modulating monocytic cell function and differentiation into macrophages. We further supported the interplay of STING1 variants and human biology by demonstrating how monocytes bearing the H232 allele were impaired in M1/M2 differentiation, interferon response and antigen presentation. Finally, we assessed the response to PD-1 inhibitor in a small cohort of melanoma patients stratified according to STING genotype. Given the contribution of the STING protein in sensing DNA viruses, bacterial pathogens and misplaced cancer DNA, these data may support the development of novel therapeutic options for infectious diseases and cancer.

Prognostic Value and Functional Consequences of Calcium Signalling in Acute Myeloid Leukaemia

Ca 2+ is a ubiquitous second messenger that regulates a large number of cellular functions by acting on various proteins and signaling pathways. Abnormal expression/function of protein involved in Ca 2+ signaling have been associated with oncogenesis. However, its implication in the mechanisms that drive normal hematopoietic cells to leukemic transformation remain largely unknown. In order to identify actors of Ca 2+ signaling of potential prognosis value in acute myeloid leukemia (AML), , we performed a systematic screening on databases and identified expression of STIM2 as negatively correlated with disease free and overall survival while ORAI2 expression presented the opposite pattern. Interestingly, we noticed that ORAI2 and STIM2 expression differed depending on FAB subtypes. Indeed the 50% of patients in the upper quartile of STIM2 expression presented a myelo-monocytic differentiation while monocytic differentiation was associated with a lower ORAI2 expression. On a series of 407 AML patients from the ALFA 07-02 protocol, we observed that higher STIM2 was found in intermediate and high ELN17 while a lower ORAI2 level was seen in ELN17 high-risk patients. We quantified STIM2 and ORAI2 expression in two AML cell lines with monocytic potential, THP1 and OCI-AML3. When cells were driven into monocytic differentiation, we observed at RNA and protein level a decreased ORAI2 expression while STIM2 increased significantly. We then focused our studies on STIM2. To decipher its function in monocytic cell lines, we chose a shRNA-mediated gene knockdown approach. We first measured the capacitive Ca 2+ entry after STIM2 knockdown and found it increased, due to a predominant downregulation of the 2.1 STIM2 isoform, which has a negative effect on Ca 2+ entry, while 2.2 STIM2 isoform was less impacted. We then performed a transcriptomic analysis using the Nanostring technology, and identified a STIM2 signature characterized by dysregulation of genes involved in apoptosis, cell cycle and chromatin modification. Phenotypically, STIM2 knockdown drastically decreased cell proliferation and viability, through activation of the mitochondrial pathway of apoptosis and blockage of cell cycle at the G2/M phase. We observed that STIM2 knockdown led to a genomic stress with DNA double strand breaks as shown by an increased p-ϒH2AX, followed by activation of the p53 pathway, decreased expression of cell cycle regulators such as CDK1-cyclin B1 and the CDC25c phosphatase, and a decreased apoptosis threshold with a low antiapoptotic/proapoptotic protein ratio. Inhibition of p53 using Pifithrin-α reverted apoptosis and cell cycle blockage whereas p-ϒH2AX induction remained high. Moreover, STIM2 knockdown led to p-ϒH2AX activation but neither apoptosis nor cell cycle blockage in the p53-deficient cell line HL60, highlighting the fact that the genomic stress occurred downstream p53 induction in cells after STIM2 knockdown. At last, using in vitro monocytic differentiation of CD34 + cells, we observed a higher STIM2 expression at RNA level in differentiated cells. Acquisition of monocytic markers was faster after STIM2 forced expression, while STIM2 knockdown impaired CD14 expression and led to a p-ϒH2AX, p53 induction, apoptosis and cell cycle blockage in agreement with data observed in leukemic cell lines. In summary, we describe here STIM2, the “forgotten” member of SOCE, as a new actor in the proliferation, survival and differentiation of human normal and malignant monocytic cells. Considering (i) the association of a high STIM2 expression with an adverse prognosis in AML and (ii) the association of STIM2 with AML harboring monocytic/myelomonocytic differentiation, STIM2 may represent an interesting protein to target in these types of leukemia in the future

The Role of Human Endogenous Retrovirus (HERV)-K119 env in THP-1 Monocytic Cell Differentiation.

Human endogenous retrovirus (HERV)-K was reportedly inserted into the human genome millions of years ago and is closely related to various diseases, including cancer and immune regulation. In our previous studies, CRISPR-Cas9-enabled knockout (KO) of the HERV-K env gene was found to potentially reduce cell proliferation, cell migration, and invasion in colorectal and ovarian cancer cell lines. The immune response involves the migration and invasion of cells and is similar to cancer; however, in certain ways, it is completely unlike cancer. Therefore, we induced HERV-K119 env gene KO in THP-1, a monocytic cell that can be differentiated into a macrophage, to investigate the role of HERV-K119 env in immune regulation. Cell migration and invasion were noted to be significantly increased in HERV-K119 env KO THP-1 cells than in MOCK, and these results were contrary to those of cancer cells. To identify the underlying mechanism of HERV-K119 env KO in THP-1 cells, transcriptome analysis and cytokine array analysis were conducted. Semaphorin7A (SEMA7A), which induces the production of cytokines in macrophages and monocytic cells and plays an important role in immune effector cell activation during an inflammatory immune response, was significantly increased in HERV-K119 env KO THP-1 cells. We also found that HERV-K119 env KO THP-1 cells expressed various macrophage-specific surface markers, suggesting that KO of HERV-K119 env triggers the differentiation of THP-1 cells from monocytic cells into macrophages. In addition, analysis of the expression of M1 and M2 macrophage markers showed that M1 macrophage marker cluster of differentiation 32 (CD32) was significantly increased in HERV-K119 env KO cells. These results suggest that HERV-K119 env is implicated in the differentiation of monocytic cells into M1 macrophages and plays important roles in the immune response.

Sox13 and M2-like leukemia-associated macrophages contribute to endogenous IL-34 caused accelerated progression of acute myeloid leukemia

Interleukin 34 (IL-34) mainly plays physiologic and pathologic roles through the sophisticated multi-ligand signaling system, macrophage colony-stimulating factor (M-CSF, CSF-1)/IL-34-CSF-1R axis, which exhibits functional redundancy, tissue-restriction and diversity. This axis is vital for the survival, differentiation and function of monocytic lineage cells and plays pathologic roles in a broad range of diseases. However, the role of IL-34 in leukemia has not been established. Here MLL-AF9 induced mouse acute myeloid leukemia (AML) model overexpressing IL-34 (MA9-IL-34) was used to explore its role in AML. MA9-IL-34 mice exhibited accelerated disease progression and short survival time with significant subcutaneous infiltration of AML cells. MA9-IL-34 cells showed increased proliferation. In vitro colony forming assays and limiting dilution transplantation experiments demonstrated that MA9-IL-34 cells had elevated leukemia stem cell (LSC) levels. Gene expression microarray analysis revealed a panel of differential expressed genes including Sex-determining region Y (SRY)-box 13 (Sox13). Furthermore, a positive correlation between the expressions of IL-34 and Sox13 was detected human datasets. Knockdown of Sox13 rescued the enhanced proliferation, high LSC level and subcutaneous infiltration in MA9-IL-34 cells. Moreover, more leukemia-associated macrophages (LAMs) were detected in MA9-IL-34 microenvironment. Additionally, those LAMs showed M2-like phenotype since they expressed high level of M2-associated genes and had attenuated phagocytic potential, suggesting that LAMs should also contribute to IL-34 caused adverse phenotypes. Therefore, our findings uncover the intrinsic and microenvironmental mechanisms of IL-34 in AML and broadens the knowledge of M-CSF/IL-34-CSF-1R axis in malignancies.

Regulation of matrix metalloproteinase-9 during monopoiesis and zinc deficiency by chromatin remodeling

IntroductionMatrix metalloproteinase-9 (MMP-9) cleaves various extracellular matrix proteins, hence significantly contributes to numerous physiological but also pathological processes. Monocytic differentiation is associated with increased MMP-9 gene expression. Interestingly, MMP-9 upregulation during monocytic differentiation is paralleled by a decline in intracellular zinc levels. Hence, an influence of zinc on the regulation of MMP-9 expression may exist. Although, previous studies suggest a vital role of zinc regarding MMP-9 activity, the possible relevance of zinc homeostasis during transcriptional regulation of MMP-9 for example via epigenetic mechanisms is rather unclear. AimThis study aims to find a correlation between zinc deficiency and MMP-9 transcriptional regulation, focusing on epigenetics as the possible mechanism behind zinc deficiency-induced changes. MethodsThe effect of differentiation and zinc deficiency on MMP-9 expression and MMP9 promoter accessibility was investigated using the acute promyelocytic cell line NB4. Intracellular free zinc levels were detected by flow cytometry. MMP-9 gene expression was measured by real-time PCR and ELISA. Analysis of chromatin structures was done using chromatin accessibility by real-time PCR (CHART) assay. ResultsDuring monocytic differentiation of NB4 cells, the decrease in intracellular zinc levels was paralleled by an increased production of MMP-9. Assessment of chromatin structure revealed increased accessibility of certain regions within the MMP-9 promoter in differentiated cells. Interestingly, upregulated activation-induced MMP-9 gene expression as well as a more accessible MMP-9 promoter were in zinc-deficient NB4 cells whereas zinc resupplementation reversed the effects. ConclusionThese data demonstrate an important role of epigenetic mechanisms in regulating MMP-9 expression under zinc deficiency. This could provide an encouraging step to expand the research on using zinc for the treatment of various pathological conditions such as inflammatory, vascular and autoimmune diseases resulting from MMP-9 deregulation.

Maitake α-glucan promotes differentiation of monocytic myeloid-derived suppressor cells into M1 macrophages

AimsMyeloid-derived suppressor cells (MDSCs) are major components of the tumor microenvironment and systemically accumulate in tumor-bearing hosts and patients with cancer, facilitating cancer progression. Maitake macromolecular α-glucan YM-2A, isolated from Grifola frondosa, inhibits tumor growth by enhancing immune responses. The present study investigated the effects of YM-2A on the immunosuppressive potential of MDSCs. Main methodsYM-2A was orally administered to CT26 tumor-bearing mice, and the number of immune cells in the spleen and tumor was measured. Splenic MDSCs isolated from the CT26 tumor-bearing mice were treated with YM-2A and co-cultured with T cells to measure their inhibitory effect on T cell proliferation. For adoptive transfer of monocytic MDSCs (M-MDSCs), YM-2A-treated M-MDSCs mixed with CT26 cells were implanted subcutaneously in the mice to measure the tumor growth rate. Key findingsYM-2A selectively reduced the accumulation of M-MDSCs but not that of polymorphonuclear MDSCs (PMN-MDSCs) in CT26 tumor-bearing mice. In tumor tissues, YM-2A treatment induced the polarity of immunostimulatory M1-phenotype; furthermore, it increased the infiltration of dendritic, natural killer, and CD4+ and CD8+ T cells. YM-2A treatment of purified M-MDSCs from CT-26 tumor-bearing mice induced dectin-1-dependent differentiation into M1 macrophages. YM-2A-treated M-MDSCs lost their inhibitory activity against proliferation and activation of CD8+ T cells. Furthermore, adoptive transfer of M-MDSCs treated with YM-2A inhibited CT26 tumor growth. SignificanceYM-2A promotes the differentiation of M-MDSCs into immunostimulatory M1 macrophages, thereby enhancing the efficacy of cancer immunotherapy.

Cytokines and HIF-1α as dysregulation factors of migration and differentiation of monocyte progenitor cells of endotheliocytes in the pathogenesis of ischemic cardiomyopathy

Background. Angiogenic endothelial dysfunction and progenitor endothelial cells (EPCs) in ischemic cardiomyopathy (ICMP) have not been studied enough.The aim. To establish the nature of changes in the cytokine profile and HIF-1α in blood and bone marrow associated with impaired differentiation of monocytic progenitor cells of endotheliocytes (CD14+VEGFR2+) in the bone marrow and their migration into the blood in patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Materials and methods. A single-stage, single-centre, observational case-control study was conducted involving 74 patients with CHD, suffering and not suffering from ICMP (30 and 44 people, respectively), and 25 healthy donors. In patients with CHD, bone marrow was obtained during coronary bypass surgery, peripheral blood – before surgery. Healthy donors were taken peripheral blood. The number of CD14+VEGFR2+ in bone marrow and blood was determined by flow cytometry; the concentration of IL-6, TNF-α, M-CSF, GM-CSF, MCP-1 and HIF-1α – by the method of enzyme immunoassay.Results. A high content of CD14+VEGFR2+ cells in the blood of patients with CHD without cardiomyopathy was established relative to patients with ICMP against the background of a comparable number of these cells in myeloid tissue. Regardless of the presence of ICMP in the blood, patients with CHD showed an excess of TNF-α, a normal concentration of IL-6, GM-CSF, HIF-1α and a deficiency of M-CSF, and in the bone marrow supernatant, the concentration of IL-6 and TNF-α exceeded that in the blood plasma (the level of GM-CSF – only in patients without cardiomyopathy). With ICMP, the normal concentration of MCP-1 was determined in the blood plasma, and with CHD without cardiomyopathy, its elevated content was determined.Conclusion. The formation of ICMP is accompanied by insufficient activation of EPCs migration with the CD14+VEGFR2+ phenotype in blood without disruption of their differentiation in the bone marrow, which associated with the absence of an increase in the concentration of MCP-1 in blood plasma and not associated with the plasma content of M-CSF, GM-CSF, HIF-1α, IL-6 and TNF-α.

The clinical and biological characterization of acute myeloid leukemia patients with S100A4 overexpression

The S100 family proteins are involved in a variety of important biological processes, most notably immune and inflammatory responses. Their dysregulation also plays a role in the pathogenesis of human cancers. S100A4, also known as metastasin, has long been regarded as a biological marker in tumor progression and metastasis in multiple solid cancers, but its clinical significance in acute myeloid leukemia (AML) has not been extensively studied. We retrospectively studied the association between S100A4 gene expression and the clinical characteristics, mutational and transcriptomic profiles of 227 AML patients treated with standard intensive chemotherapy. Genetic mutations of myeloid disease associated genes were analyzed by Sanger sequencing. Microarray-based transcriptomic gene expression profiling was performed on archived bone marrow mononuclear cells. Bioinformatic analyses, including differential gene expression and gene set enrichment analysis, were conducted to delineate the underlying pathogenic mechanisms. Higher S100A4 expression was associated with older age, monocytic differentiation of leukemic cells, and adverse clinical outcome. S100A4 high-expressors had inferior overall survival and disease-free survival; this finding could be validated in the TCGA AML cohort (both the microarray and RNA-seq platforms). Multivariate Cox regression analysis supported S100A4 as an independent prognostic factor. Bioinformatic analysis showed that AML with higher S100A4 expression was enriched for the interferon, NLRP3 inflammasome, and epithelial-mesenchymal transition pathways. This study provides evidence that S100A4 overexpression serves as a poor prognostic biomarker in AML, holds potential to guide treatment planning in the clinic, and indicates novel therapeutic directions.

Distinct metabolic states guide maturation of inflammatory and tolerogenic dendritic cells

Cellular metabolism underpins immune cell functionality, yet our understanding of metabolic influences in human dendritic cell biology and their ability to orchestrate immune responses is poorly developed. Here, we map single-cell metabolic states and immune profiles of inflammatory and tolerogenic monocytic dendritic cells using recently developed multiparametric approaches. Single-cell metabolic pathway activation scores reveal simultaneous engagement of multiple metabolic pathways in distinct monocytic dendritic cell differentiation stages. GM-CSF/IL4-induce rapid reprogramming of glycolytic monocytes and transient co-activation of mitochondrial pathways followed by TLR4-dependent maturation of dendritic cells. Skewing of the mTOR:AMPK phosphorylation balance and upregulation of OXPHOS, glycolytic and fatty acid oxidation metabolism underpin metabolic hyperactivity and an immunosuppressive phenotype of tolerogenic dendritic cells, which exhibit maturation-resistance and a de-differentiated immune phenotype marked by unique immunoregulatory receptor signatures. This single-cell dataset provides important insights into metabolic pathways impacting the immune profiles of human dendritic cells.

The immunoreceptor SLAMF8 promotes the differentiation of follicular dendritic cell-dependent monocytic cells with B cell-activating ability.

Follicular dendritic cells (FDCs) play a crucial role in generating high-affinity antibody-producing B cells during the germinal center (GC) reaction. Herein, we analysed the altered gene expression profile of a mouse FDC line, FL-Y, following lymphotoxin β receptor stimulation, and observed increased Slam-family member 8 (Slamf8) mRNA expression. Forced Slamf8 expression and SLAMF8-Fc addition enhanced the ability of FL-Y cells to induce FDC-induced monocytic cell (FDMC) differentiation. FDMCs accelerated GC-phenotype proliferation in cultured B cells, suggesting that they are capable of promoting GC responses. Furthermore, a pulldown assay showed that SLAMF8-Fc could bind to SLAMF8-His. Overall, the homophilic interaction of SLAMF8 promotes FDMC differentiation and SLAMF8 might act as a novel regulator of GC responses by regulating FDMC differentiation.

Vitamin D Derivatives in Acute Myeloid Leukemia: The Matter of Selecting the Right Targets.

Acute myeloid leukemia (AML) is an aggressive and often fatal hematopoietic malignancy. A very attractive way to treat myeloid leukemia, called “differentiation therapy”, was proposed when in vitro studies showed that some compounds are capable of inducing differentiation of AML cell lines. One of the differentiation-inducing agents, all-trans-retinoic acid (ATRA), which can induce granulocytic differentiation in AML cell lines, has been introduced into clinics to treat patients with acute promyelocytic leukemia (APL) in which a PML-RARA fusion protein is generated by a chromosomal translocation. ATRA has greatly improved the treatment of APL. Since 1,25-dihydroxyvitamin D (1,25D) is capable of inducing monocytic differentiation of leukemic cells, the idea of treating other AMLs with vitamin D analogs was widely accepted. However, early clinical trials in which cancer patients were treated either with 1,25D or with analogs did not lead to conclusive results. Recent results have shown that AML types with certain mutations, such as isocitrate dehydrogenase (IDH) mutations, may be the right targets for differentiation therapy using 1,25D, due to upregulation of vitamin D receptor (VDR) pathway.

The Promoter of the Immune-Modulating Gene TIR-Containing Protein C of the Uropathogenic Escherichia coli Strain CFT073 Reacts to the Pathogen's Environment.

The TIR-containing protein C (TcpC) of the uropathogenic Escherichia coli strain CFT073 modulates innate immunity by interfering with the Toll-like receptor and NALP3 inflammasome signaling cascade. During a urinary tract infection the pathogen encounters epithelial and innate immune cells and replicates by several orders of magnitude. We therefore analyzed whether these cell types and also the density of the pathogen would induce the recently defined promoter of the CFT073 tcpC gene to, in time, dampen innate immune responses. Using reporter constructs we found that the uroepithelial cell line T24/83 and the monocytic cell line THP-1 induced the tcpC promoter. Differentiation of monocytic THP-1 cells to macrophages increased their potential to switch on the promoter. Cell-associated CFT073 displayed the highest promoter activity. Since potassium represents the most abundant intracellular ion and is secreted to induce the NLRP3 inflammasome, we tested its ability to activate the tcpC promoter. Potassium induced the promoter with high efficiency. Sodium, which is enriched in the renal cortex generating an antibacterial hypersalinity, also induced the tcpC promoter. Finally, the bacterial density modulated the tcpC promoter activity. In the search for promoter-regulating proteins, we found that the DNA-binding protein H-NS dampens the promoter activity. Taken together, different cell types and salts, present in the kidney, are able to induce the tcpC promoter and might explain the mechanism of TcpC induction during a kidney infection with uropathogenic E. coli strains.

Inhibiting BTK and IDO Enhances Dendritic Cell-Mediated Immune Response.

Dual inhibition of BTK and IDO promotes monocytic lineage-derived dendritic cell differentiation.

Linking GATA2 Deficiency to Dysregulated Innate Immune Signaling

Cell type-specific transcription factors governing hematopoietic stem and progenitor cell transitions establish networks containing hundreds of genes and proteins. Network complexity renders it challenging to discover essential versus modulatory or redundant components. This scenario is exemplified by GATA2 mechanisms that control hematopoiesis during embryogenesis. Loss of a far upstream Gata2 enhancer (-77) disrupts the GATA2-dependent genetic network governing hematopoietic progenitor cell differentiation (Johnson KD. et al., Sci. Adv., 2015). The aberrant network includes the transcription factor Interferon Regulatory Factor-8 and a host of innate immune regulators, including Toll-like receptors (TLRs) (Johnson KD. et al., J. Exp. Med., 2020). Mutant embryonic progenitors lose the capacity to balance production of diverse hematopoietic progeny and generate excessive monocytic progeny. As IRF8 is vitally important for monocytic and dendritic cell differentiation (Yanez A. and Goodridge H., Curr. Opin. Hematol., 2016), we asked whether IRF8 is essential, contributory, or inconsequential. Using a double-mutant genetic rescue in vivo system, we demonstrated that reducing Irf8, in the context of the -77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell-committed progenitors. In -77 -/- E14.5 fetal livers, monocyte progenitors (MPs) increased 2.3-fold (P = 0.006), granulocyte progenitors (GPs) decreased 2.2-fold (P = 0.003) and common dendritic cell progenitors (CDPs) increased 10.2-fold (P = 0.021) relative to wildtype littermates. Ablating Irf8 in -77 mutants (-77 -/-; Irf8-/-) restored MPs and CDPs to wildtype levels and reversed the GP deficiency; further increasing GPs 4.2-fold relative to wildtype (P = 0.0009). Despite many dysregulated components that control vital transcriptional, signaling and immune processes, the aberrant elevation of a single transcription factor deconstructed the embryonic hematopoiesis program. We analyzed the mechanistic and biological implications of IRF8 dysregulation concomitant with ectopic upregulation of other innate immune genes (including Toll-like receptors (TLRs) in GATA2-deficient embryonic progenitors. In principle, such genes might function upstream, downstream, or in parallel with IRF8. Based on TLR upregulation and TLR roles in progenitor mechanisms (Nagai Y. et al., Immunity, 2006; Schuettpelz L. et al., Leukemia, 2014; Caiado F. et al., J. Exp. Med., 2021), we tested whether GATA2 deficiency in embryonic progenitors impacts cellular responsiveness to TLR ligands. Wild type and -77 enhancer-mutant progenitors were treated with increasing concentrations of the TLR1/2 ligand Pam 3CSK 4. The mutant progenitors were hypersensitive to Pam 3CSK 4, which resulted in supra-physiological induction of Tnf expression (2.8-fold at 34 nM, P = 0.004; 3.2-fold at 68 nM, P = 0.0003). Quantitative analyses indicated that hypersensitivity reflected increased Pam 3CSK 4 efficacy, but not potency. GATA2 re-expression in the mutant progenitors attenuated the elevated IRF8 expression and TLR signaling, normalizing Tnf and Ccl3 expression to a level comparable to that of wild type progenitors. In GATA2-rescued mutant progenitors, Tnf and Ccl3 expression decreased 3.9-fold (P = 0.005) and 2.5-fold (P = 0.047), respectively. Thus, GATA2 suppresses TLR signaling in embryonic progenitors. Ongoing studies are elucidating the mechanistic interconnections between IRF8- and TLR-dependent inflammatory networks in GATA2 deficiency during embryonic and adult hematopoiesis in cell populations and single cells, relationships between murine and human mechanisms, and the impact of targeted interventions that modulate these mechanisms. DisclosuresNo relevant conflicts of interest to declare.

1030TiP An open-label multicenter phase Ib study of AN0025, a selective inhibitor of the prostaglandin E2 receptor-type-4 receptor, in combination with pembrolizumab in patients with advanced solid tumors

AN0025 is a selective inhibitor of the prostaglandin E2 type-4 receptor, inhibiting PGE2-mediated differentiation and suppressive function of monocytic myeloid cells, which contribute to the immunosuppressive tumor microenvironment. These cells are associated with a poorer prognosis in many cancers. A previous Ph 1 study with AN0025 was conducted to assess safety, MTD and RP2D. AN0025 was well tolerated in 30 patients with advanced cancer at 125, 250, 500, 750 mg QD with no DLTs. The MTD was not reached.

The Thymidine Dideoxynucleoside Analogue, Alovudine, Inhibits the Mitochondrial DNA Polyermase, POLG, Impairs Oxidative Phosphorylation and Promotes Monocytic Differentiation in AML

Mitochondria contain circular DNA that encodes for 13 proteins that comprise subunits of the respiratory chain and are necessary for oxidative phosphorylation. The replication of mitochondrial DNA (mt DNA) requires nucleotides from both the mitochondria and cytoplasm and is regulated by POLG (Polymerase γ), the sole mtDNA polymerase. Acute Myeloid Leukemia (AML) cells and stem cells have increased mitochondrial biogenesis and increased reliance on oxidative phosphorylation. Moreover, AML cells over-express cytoplasmic nucleoside kinases that phosphorylate nucleosides to their active forms. Alovudine (3‘-deoxy-3‘-fluorothymidine) is a dideoxynucleoside analogue of thymidine that is phosphorylated by nucleoside kinases into its active form where it selectively and potently inhibits POLG over nuclear polymerase. Therefore, we evaluated the effects of alovudine on AML cells in vitro and in vivo.OCI-AML2 and MV4-11 leukemia cells were treated with increasing concentrations of alovudine. After 6 days of treatment, alovudine decreased mtDNA by over 95% in OCI-AML2 and MV4-11 cells at 1000nM and 50nM, respectively. In contrast, cytarabine which inhibits nuclear DNA replication, did not alter levels of mtDNA. We measured levels of mt-encoded COXI and COXII proteins, subunits of respiratory chain subunit IV. Concentrations of alovudine that reduced mtDNA also reduced levels of COXI and COXII proteins in OCI-AML2 and MV4-11 cells. In contrast, alovudine did not change levels of COXIV, a subunit of respiratory chain complex IV, which is encoded by nuclear DNA, translated in the cytoplasm, and imported into the mitochondria. Consistent with reductions in respiratory chain IV subunits, alovudine decreased basal oxygen consumption in OCI-AML2 and MV4-11 cells. Finally, concentrations of alovudine that reduced mtDNA and impaired oxidative phosphorylation reduced the growth and viability of AML cell lines.We also tested the effects of alovudine on primary AML cell; In primary AML cells alovudine reduced mtDNA and decreased cell viability.Next, we examined the effects of alovudine on the growth of AML cells in vivo . SCID mice xenografted with OCI-AML2 cells were treated with alovudine (50mg/kg p.o bid 5/7 days). Alovudine decreased the growth of AML cells in vivo by 70%. At doses that decreased AML growth, no change in mouse body weight, serum chemistries, or organ histology was observed.Recent studies demonstrate that mitochondrial pathways and metabolism can regulate cell fate and differentiation. Therefore, we explored the effects of alovudine on the differentiation of OCI-AML2, MV4-11and TEX cell-lines, and the 8277 primary AML culture system. Alovudine upregulated expression of CD11b, CD14 and CD15 surface markers associated with cell differentiation. In addition, alovudine increased non-specific esterase staining and induced monocytic morphology.In summary, aluvodine is a potent inhibitor of POLG that impairs oxidative phosphorylation in AML and decreases the growth of AML cells in vitro and in vivo . We discovered that inhibition of POLG induces the monocytic differentiation of AML cells. Thus, we identified new mechanisms by which mitochondrial pathways control differentiation in AML and highlight the POLG inhibitor, aluvodine, as a novel potential therapeutic agent for this disease DisclosuresSchimmer:Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria. Rizoska:Medivir: Employment. Targett-Adams:Medivir: Employment. Albertella:Medivir: Employment.

Myosin Phosphatase Is Implicated in the Control of THP-1 Monocyte to Macrophage Differentiation.

Monocyte to macrophage differentiation is characterized by the activation of various signal transduction pathways, which may be modulated by protein phosphorylation; however, the impact of protein kinases and phosphatases is not well understood yet. It has been demonstrated that actomyosin rearrangement during macrophage differentiation is dependent on Rho-associated protein kinase (ROCK). Myosin phosphatase (MP) target subunit-1 (MYPT1) is one of the major cellular substrates of ROCK, and MP is often a counter enzyme of ROCK; therefore, MP may also control macrophage differentiation. Changes in MP activity and the effects of MP activation were studied on PMA or l,25(OH)2D3-induced differentiation of monocytic THP-1 cells. During macrophage differentiation, phosphorylation of MYPT1 at Thr696 and Thr853 increased significantly, resulting in inhibition of MP. The ROCK inhibitor H1152 and the MP activator epigallocatechin-3-gallate (EGCG) attenuated MYPT1 phosphorylation and concomitantly decreased the extent of phosphorylation of 20 kDa myosin light chain. H1152 and EGCG pretreatment also suppressed the expression of CD11b and weakened the PMA-induced adherence of the cells. Our results indicate that MP activation/inhibition contributes to the efficacy of monocyte to macrophage differentiation, and this enzyme may be a target for pharmacological interventions in the control of disease states that are affected by excessive macrophage differentiation.