Apoptosis In Myeloid Cells Research Articles

Peds1 deficiency in zebrafish results in myeloid cell apoptosis and exacerbated inflammation

Plasmalogens are glycerophospholipids with a vinyl ether bond that confers unique properties. Recent identification of the gene encoding PEDS1, the desaturase generating the vinyl ether bond, enables evaluation of the role of plasmalogens in health and disease. Here, we report that Peds1-deficient zebrafish larvae display delayed development, increased basal inflammation, normal hematopoietic stem and progenitor cell emergence, and cell-autonomous myeloid cell apoptosis. In a sterile acute inflammation model, Peds1-deficient larvae exhibited impaired inflammation resolution and tissue regeneration, increased interleukin-1β and NF-κB expression, and elevated ROS levels at the wound site. Abnormal immune cell recruitment, neutrophil persistence, and fewer but predominantly pro-inflammatory macrophages were observed. Chronic skin inflammation worsened in Peds1-deficient larvae but was mitigated by exogenous plasmalogen, which also alleviated hyper-susceptibility to bacterial infection, as did pharmacological inhibition of caspase-3 and colony-stimulating factor 3-induced myelopoiesis. Overall, our results highlight an important role for plasmalogens in myeloid cell biology and inflammation.

Single-cell landscape revealed immune characteristics associated with disease phases in brucellosis patients.

A comprehensive immune landscape for Brucella infection is crucial for developing new treatments for brucellosis. Here, we utilized single-cell RNA sequencing (scRNA-seq) of 290,369 cells from 35 individuals, including 29 brucellosis patients from acute (n = 10), sub-acute (n = 9), and chronic (n = 10) phases as well as six healthy donors. Enzyme-linked immunosorbent assays were applied for validation within this cohort. Brucella infection caused a significant change in the composition of peripheral immune cells and inflammation was a key feature of brucellosis. Acute patients are characterized by potential cytokine storms resulting from systemic upregulation of S100A8/A9, primarily due to classical monocytes. Cytokine storm may be mediated by activating S100A8/A9-TLR4-MyD88 signaling pathway. Moreover, monocytic myeloid-derived suppressor cells were the probable contributors to immune paralysis in acute patients. Chronic patients are characterized by a dysregulated Th1 response, marked by reduced expression of IFN-γ and Th1 signatures as well as a high exhausted state. Additionally, Brucella infection can suppress apoptosis in myeloid cells (e.g., mDCs, classical monocytes), inhibit antigen presentation in professional antigen-presenting cells (APCs; e.g., mDC) and nonprofessional APCs (e.g., monocytes), and induce exhaustion in CD8+ T/NK cells, potentially resulting in the establishment of chronic infection. Overall, our study systemically deciphered the coordinated immune responses of Brucella at different phases of the infection, which facilitated a full understanding of the immunopathogenesis of brucellosis and may aid the development of new effective therapeutic strategies, especially for those with chronic infection.

Methylene blue dose-dependently induces cutaneous inflammation and heat hyperalgesia in a novel rat model.

Methylene blue (MB) has been shown to reduce mortality and morbidity in vasoplegic patients after cardiac surgery. Though MB is considered to be safe, extravasation of MB leading to cutaneous toxicity has been reported. In this study, we sought to characterize MB-induced cutaneous toxicity and investigate the underlying mechanisms. To induce MB-induced cutaneous toxicity, we injected 64 adult male Sprague-Dawley rates with 200 µL saline (vehicle) or 1%, 0.1%, or 0.01% MB in the plantar hind paws. Paw swelling, skin histologic changes, and heat and mechanical hyperalgesia were measured. Injection of 1%, but not 0.1% or 0.01% MB, produced significant paw swelling compared to saline. Injection of 1% MB produced heat hyperalgesia but not mechanical hyperalgesia. Pain behaviors were unchanged following injections of 0.1% or 0.01% MB. Global transcriptomic analysis by RNAseq identified 117 differentially expressed genes (111 upregulated, 6 downregulated). Ingenuity Pathway Analysis showed an increased quantity of leukocytes, increased lipids, and decreased apoptosis of myeloid cells and phagocytes with activation of IL-1β and Fos as the two major regulatory hubs. qPCR showed a 16-fold increase in IL-6 mRNA. Thus, using a novel rat model of MB-induced cutaneous toxicity, we show that infiltration of 1% MB into cutaneous tissue causes a dose-dependent pro-inflammatory response, highlighting potential roles of IL-6, IL-1β, and Fos. Thus, anesthesiologists should administer dilute MB intravenously through peripheral venous catheters. Higher concentrations of MB (1%) should be administered through a central venous catheter to minimize the risk of cutaneous toxicity.

Programmable DNA hydrogel provides suitable microenvironment for enhancing autophagy-based therapies in intervertebral disc degeneration treatment

The pathogenesis of intervertebral disc degeneration (IVDD) is attributed to metabolic dysregulation within the extracellular matrix and heightened apoptosis of nucleus pulposus cells (NPC). Therefore, a potential therapeutic strategy for managing IVDD involves the reestablishment of metabolic equilibrium within the extracellular matrix and the suppression of excessive myeloid cell apoptosis. The microRNA, miR-5590, displays marked differential expression in degenerative nucleus pulposus (NP) tissues and exerts a direct influence on the regulation of DDX5 expression. This, in turn, modulates mammalian target of rapamycin (mTOR) phosphorylation, thereby impacting autophagy and apoptosis. However, ensuring the smooth delivery of miRNA to a specific injury site poses a significant challenge. To address this issue, a multifunctional DNA hydrogel was developed and subsequently loaded with miR-5590 via spherical nucleic acids (SNAs) for the treatment of IVDD. The hydrogel, which exhibits versatility, has the potential to be administered through injection at the site of injury, resulting in a consistent and prolonged release of miR-5590. This leads to the creation of a genetic microenvironment within the NP, which triggers the onset of autophagy in NPCs and subsequently suppresses apoptosis. As a result, this process regulates the metabolic equilibrium within the extracellular matrix, thereby impeding the in vitro and in vivo progression of IVDD. The amalgamation of miRNAs and biomaterials offers a promising therapeutic strategy for the management of IVDD in clinical settings.

Kidney medullary sodium chloride concentrations induce neutrophil and monocyte extracellular DNA traps that defend against pyelonephritis in vivo

Urinary tract infections are common. Here, we delineate a role of extracellular DNA trap (ET) formation in kidney antibacterial defense and determine mechanisms of their formation in the hyperosmotic environment of the kidney medulla. ET of granulocytic and monocytic origin were present in the kidneys of patients with pyelonephritis along with systemically elevated citrullinated histone levels. Inhibition of the transcription coregulatory, peptidylarginine deaminase 4 (PAD4), required for ET formation, prevented kidney ET formation and promoted pyelonephritis in mice. ETs predominantly accumulated in the kidney medulla. The role of medullary sodium chloride and urea concentrations in ET formation was then investigated. Medullary-range sodium chloride, but not urea, dose-, time- and PAD4-dependently induced ET formation even in the absence of other stimuli. Moderately elevated sodium chloride promoted myeloid cell apoptosis. Sodium gluconate also promoted cell death, proposing a role for sodium ions in this process. Sodium chloride induced myeloid cell calcium influx. Calcium ion-free media or -chelation reduced sodium chloride-induced apoptosis and ET formation while bacterial lipopolysaccharide amplified it. Autologous serum improved bacterial killing in the presence of sodium chloride-induced ET. Depletion of the kidney sodium chloride gradient by loop diuretic therapy diminished kidney medullary ET formation and increased pyelonephritis severity. Thus, our data demonstrate that ETs may protect the kidney against ascending uropathogenic E.coli and delineate kidney medullary range sodium chloride concentrations as novel inducers of programmed myeloid cell death.

Comment on: Detection of a rare variant in PSTPIP1 through three generations in a family with an initial diagnosis of FMF/MKD-overlapping phenotype.

Dear Editor, We read with great interest the article by Özkılınç Önen et al. that was recently published in Rheumatology [1]. In this impressive article, the authors focused on an interesting issue regarding the genetic puzzle presented in four members of three generations of a Turkish family, with an FMF/mevalonate kinase deficiency (MKD)-overlapping phenotype, who did not carry any MEFV/MVK pathogenic variants. The authors of this article by an elegant approach refined the genetic heterogeneity observed in these patients, by demonstrating the presence of a p. Arg228Cys PSTPIP1 variant in the FMF/MKD phenotype. Notably, PSTPIP1 variants had been also described previously in cases with pyogenic arthritis, pyoderma gangrenosum and Pyogenic Arthritis, Pyoderma gangrenosum and Acne (PAPA) [1]. The results of the aforementioned study [1] contribute significantly in the long-standing, challenging question of the genetic aetiology of FMF, an autosomal, single-gene, recessively inherited disease, characterized by acute, self-limited attacks of fever, serositis and abdominal pain, without any apparent trigger or autoimmune aetiology [2]. The MEFV gene, causing FMF, encodes the protein ‘pyrin’. Considering that no diagnostic biochemical markers are available in FMF thus far, detection of MEFV mutations is a very important issue for an early and correct disease diagnosis. However, the presence of a considerable proportion of FMF cases without MEFV pathogenic variants as well as the high number of heterozygote patients with typical symptoms of the disease have driven a number of alternative aetiopathogenic hypotheses. To this end, our group attempted to elucidate the role of the pyrin-interacting [3], pro-apoptotic protein Siva as a putative mediator of the FMF development. Thus, we hypothesized that mutations in the gene encoding pyrin (MEFV) may affect Siva-mediated apoptosis. Although Siva protein binds pyrin, as shown by transfection and immunoprecipitation experiments, no differences in rates of apoptosis in myeloid cells (THP-1) upon transfection with either wt pyrin or mutant forms of pyrin were observed as assessed by FACS and Western blotting [4]. Therefore, we did not manage to confirm our hypothesis, which would have demonstrated for the first time a connection between apoptosis and inflammation, considering that our experimental results suggested that Siva-mediated unprovoked apoptosis is not likely to be involved in FMF pathogenesis [4]. Moreover, aiming to refine further the molecular basis of FMF, we proceeded in the MEFV knockdown in human myelomonocytic cells in an attempt to identify deregulated microRNAs. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that the identified miR-4520a targets genes are implicated in autophagy through regulation of Ras homolog enriched in brain/mammalian target of rapamycin (RHEB/mTOR) signalling, thus demonstrating for the first time the role of deregulated autophagy in the pathogenesis of FMF [5].

PECTIN MODULATES HEMATO-BIOCHEMICAL PARAMETER, HISTOPATHOLOGY, OXIDATIVE STRESS BIOMARKERS, CYTOKINES AND EXPRESSION OF HEPCIDN GENE IN LEAD INTOXICATED RATS

Publications concerning the protective effect of pectin against lead induced toxicity in rats are not available. In order to study such effect, 40 male rats were divided into 3 groups. The first group was contained 10 rats that kept as control group. The second group was contained 10 rats that received pectin at dose of 100 mg/kg BW during experimental period (8 weeks). The third group was contained 20 rats that received 400mg/kg BW of lead acetate daily for 4 weeks then divided into two subgroups (3A and 3B). Subgroup 3A contained 10 rats that still receive lead acetate in the same dosage whereas, subgroup B co-treated with lead acetate and pectin daily for another 4 weeks. Blood samples were collected after 2, 4 and 8 weeks from the start of the experiment. Liver, kidney and bone marrows were collected only at the end of the experiment. Lead acetate induced anemia only after 4 weeks of administration as reflected on decreased values of Hb, PCV, MCV, MCH and MCHC. These indices remained at lower levels in lead acetate treated groups until the end of the experiment. Concentrations of serum ferritin, iron, total antioxidant capacity (TAC) and reduced glutathione (GSH) and the expression of hepatic hepcidin gene were decreased significantly in lead acetate intoxicated rats compared to control. Activities of ALT and AST and concentrations of urea, creatinine, Nitric oxide (NO), TNF-α, IL-6, total iron binding capacity (TIBC) and lead were increased significantly in lead acetate intoxicated group compared to control. Hepatic degeneration and hemorrhage, renal lytic necrosis and apoptosis of myeloid cells were most prominent changes in lead intoxicated rats. Lead acetated related changes were improved by co-treatment with pectin however; normal control values have not been achieved. Conclusively, pectin is recommended to protect against lead acetate toxicity in rats. Key words: lead acetate; toxicity; pectin; hepcidin; oxidative stress biomarkers; histopathology

Abstract 5610: Targeting Rb1 in experimental pancreatic cancer remodels immune cell landscape and suppresses tumor progression

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) remains among the cancers with the poorest prognosis. Currently, no effective treatment available for PDA. Immunotherapy that has been shown effective for several cancer types is not successful for PDA treatment overall. The possible reasons of this PDA immunotherapy resistance include lack of neoantigen mutations, low T cell responses and overall immunosuppression state in the tumor microenvironment mediated by myeloid cells and tumor stroma. Recent studies suggest that targeting tumor microenvironment can overcome this immunotherapy resistance and significantly improve the disease outcome. Retinoblastoma (Rb1) protein is a known tumor suppressor protein. In addition, there is accumulating evidence for its role in immune cell response as well, including regulation of apoptosis in myeloid cells. However, the mechanisms of this phenomenon remain not clear. Here we show that targeting Rb1 with a novel molecule AP-3-84 developed in our lab causes apoptosis of tumor-associated macrophages (TAMs) in the model of PDA, induces T cell infiltration of the tumor and decreases tumor burden. Results: Recently our lab has developed the new small molecule AP-3-84 that is able to bind Rb1 and modulate its activity. Originally, we found that this Rb1 modulator can induce cell death of TAMs and thioglycolate-induced macrophages, but not of the other cell types (neither tumor nor T cells). Gene expression changes upon AP-3-84 treatment showed the induction of oxidative and ER stress programs with a clear activation of p53-dependent genes and caspases. We tested the use of AP-3-84 as immunotherapy against PDA cancer growth using 2 PDA cell lines intrinsically characterized by low T cell and high T cell infiltration (T-low-PDA and T-high-PDA, respectively, originally generated and characterized by J. Li et al., Immunity 2018). In accordance with our in vitro observation, AP-3-84 was able to reshape immune cell landscape in mice with T-high-PDA by depleting macrophages and inducing T cell infiltration. Importantly, those changes were accompanied by significant reduction of tumor growth. Addition of AP-3-84 to immunotherapy (anti-CD40/PD1/CTL4) was also able to decrease tumor burden in T-low-PDA model, which is otherwise resistant to this immunotherapy. Thus, AP-3-84 treatment was able to significantly improve the outcome of experimental PDA by altering immune cell subsets and represent a prospective clinical approach for the clinic. Statistical analysis of the data was conducted using unpaired Student t-test with additional false discovery rate adjustment for multiple comparisons for gene expression analysis. Conclusions: Modulation of Rb1 in myeloid cells is a novel promising target for PDA treatment. This approach reduces PDA tumor burden by shifting the immune cell landscape towards greater T cell infiltration upon tumor macrophage depletion. Citation Format: Evgenii N. Tcyganov, Taekyoung Kwak, Adi Narayana Reddy Poli, Laxminarasimha Donthireddy, Joel Cassel, Andrew Kossenkov, Rahul Shinde, Ben Z. Stanger, Joseph Salvino, Luis J. Montaner. Targeting Rb1 in experimental pancreatic cancer remodels immune cell landscape and suppresses tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5610.

Tumor Suppressor, NORE1A (RASSF5), Is an Icsbp-Dependent Inhibitor of Aberrant Stress Granulopoiesis

Within the innate immune system, emergency (stress) granulopoiesis serves the specific and essential function of periodic granulocyte production in response to an infectious or inflammatory challenge. Emergency granulopoiesis occurs in four phases (immediate release of mature granulocytes from the bone marrow, expansion of the common granulocyte/monocyte progenitor pool, accelerated differentiation, and termination of the response) and requires IL1β, an IL1β-dependent increase in G-CSF and the transcription factors Stat3 and C/EPBβ. Our group has previously shown that interferon consensus sequence binding protein (Icsbp) is required for the termination of emergency granulopoiesis. Icsbp deficiency is associated with increased and sustained production of Fap1 and Gas2, aberrantly sustained granulocytosis and promotion of myeloid leukemia. We have identified a novel Icsbp target gene, NORE1A (RASSF5), that is likewise required for the termination of emergency granulopoiesis. Nore1A is a tumor suppressor that is down-regulated in many human cancers. We found that Icsbp acts as a NORE1A transcriptional activator and that Nore1A expression is decreased in bone marrow-derived myeloid progenitor cells from ICSBP -/- mice compared to wild-type (WT), and does not appropriately increase during ex vivo differentiation with G-CSF or IL1β. In cell culture, ICSBP -/- murine bone marrow cells were resistant to intrinsic or Fas-induced apoptosis compared to WT. However, transduction with a Nore1A expression vector rescued Fas-induced apoptosis in ICSBP -/- murine bone marrow-derived myeloid progenitor cells, associated with activation of the pro-apoptotic Mst1 kinase. We injected WT or NORE1 -/- mice intraperitoneally every 4 weeks with an ovalbumin/alum mixture (to stimulate emergency granulopoiesis) or saline control. NORE1 -/- mice demonstrated aberrantly exaggerated granulocytosis in response to repeated cycles of ovalbumin/alum stimulation compared to WT mice. NORE1 -/- mice also demonstrated a tendency to develop myeloid leukemia, even in the absence of overt immune stimulation. These findings suggest that Nore1A inhibits aberrant stress granulopoiesis by promoting apoptosis of myeloid cells in an Icsbp-dependent fashion. Our data implicate Nore1A deficiency in the deregulation of granulopoiesis and promotion of leukemogenesis that accompanies clinical scenarios associated with decreased Icsbp activity, such as human chronic myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

JAGN1 mutations in severe congenital neutropenia.

Severe congenital neutropenia (SCN) was first clinically described in 1956 by Swedish pediatrician Rolf Kostmann, who recognized that infants who suffered from severe bacterial infections with absolute neutrophil counts (ANC) persistently below 0.5 × 109/l represented an inherited genetic disorder.1 SCN is now known to be caused by mutations in at least 25 genes and has autosomal recessive (AR), autosomal dominant (AD) and X-linked forms.2 The most common cause is due to AD mutations in the neutrophil elastase gene, ELANE, while the original disease Kostmann described was found to be due to AR mutations in HAX1.3, 4 Many of the genes causing SCN appear to be involved in endoplasmic reticulum (ER) or mitochondrial function and their disruption is involved in increasing ER stress and enhancing neutrophil apoptosis. In this issue the paper by Khandagale et al.5 whole exome sequencing techniques were applied to 27 AR SCN patients and three were found to have homozygous mutations in jagunal homolog 1 (JAGN1) which was first described as a cause of SCN in 2014 by Boztug et al.6 Jagunal was originally described as being critical for oocyte development in Drosophila via its role in ER reorganization.7 This new study further enhances our knowledge of how JAGN1 mutations can lead to ER stress and intracellular calcium activation of calpain that leads to apoptosis of myeloid cells. The experiments reported here utilized patient neutrophils, human bone marrow and the HL-60 cell line (derived from a human acute promyelocytic leukemia) to demonstrate aberrant neutrophil granules and a predilection to higher cytosolic calcium upon stimulation and enhanced calpain-dependent apoptosis in the presence of the mutant JAGN1 which could be blocked by addition of a specific inhibitor (PD150606) or by silencing JAGN1. It is of note that knocking down JAGN1 with small interfering (si)-RNA did not affect susceptibility of HL-60 cells to cell death, suggesting a dominant pathophysiologic effect of patient mutant JAGN1 on a calpain-mediated process despite an autosomal recessive pattern of inheritance. Khandagale et al. have achieved a very important breakthrough in understanding how JAGN1 mutations promote a non-apoptotic, calpain-dependent form of cell death. The authors acknowledge that further work will be needed to fully understand this pathway. The mainstay of treatment for SCN has been filgrastim since its approval by the FDA in 1991.8 It is a recombinant human granulocyte-stimulating factor that must be injected. While improving quality of life and survival by enhancing numbers and function of neutrophils in most types of SCN, long-term use has been associated with patients being at increasing risk of clonal myeloid disorders and malignancies.9 Whether this is a direct toxicity or due to prolonged survival of the patients allowing evolution of malignant clones from the prolonged pathophysiologic stress from effects of the underlying mutation remains controversial. Other experimental treatments may be appropriate in carefully selected SCN patients such as small-molecule CXCR4 inhibitors such as plerixafor or mavorixafor for WHIM syndrome,10 empagliflozin for G6PT deficiency,11 and perhaps neutrophil elastase inhibitors such as alvelestat for ELANE mutations or JAGN1 inhibitors for mutant JAGN1. Bone marrow transplantation can cure SCN, but is limited by the availability of suitable donors, and is associated with the risk of graft-versus-host disease, infectious mortality and other complications which will be improved by utilizing more specific conditioning regimens.12 Donor availability has been made less of a concern since haploidentical transplant has become more common. Genetic correction of neutrophil gene defects has already been successfully undertaken13 and may be even more practical in the future. In AD SCN, recent evidence suggests deletion of the disease allele may be a simple and effective technique without need to specifically correct the gene defect. In the case of WHIM syndrome, evidence suggests that removal of one copy of CXCR4 enhances haematopoietic stem cell engraftment and survival without the need for conditioning.14 Similarly, editing and removal of ELANE may be curative in the future.15

MNDA controls the expression of MCL-1 and BCL-2 in chronic lymphocytic leukemia cells

The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course.

Molecular Targeting and Rational Chemotherapy in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a molecularly complex disease with multiple aberrant genetic pathways involved in its pathogenesis. Approximately one-third to one-half of patients with AML would relapse, and no standard therapy is established for relapsing and/or refractory AML (RR-AML) yet. It is unlikely that blockage of only one specific pathway will lead to prolonged remissions and cures in all fractions of the AML patients population. Nowadays, novel therapeutic agents with rational combination are being recognized which improve the cure rate for relapsed AML. These drugs and their metabolites impart unique properties in the interaction with each of the intracellular targets and metabolic enzymes whereby resulting in unique clinical activity. To date, most of the combinations have used a targeted agent combined with standard agents such as anthracyclines, cytarabine, or hypomethylating agents to improve the outcome. Rational combinations of DNA damage-inducing therapies with DNA methyltransferase and histone deacetylase inhibitors synergistically enhance the DNA damage, growth inhibition and apoptosis of myeloid cells. This review makes a thorough look at current antineoplastic agents for AML with emphasis on its genetics and molecular mechanisms of action and the role of combination regimens.

Effects of triptolide and BET protein inhibitor JQ1 on the proliferation and apoptosis of MLL-rearranged acute myeloid leukemia cells and their mechanisms

Objective To investigate the effects of triptolide (TPL) and BET protein inhibitor JQ1 on proliferation inhibition and apoptosis induction of MLL-rearranged acute myeloid leukemia (AML) cell line MV4-11, and to explore their synergistic mechanisms. Methods MV4-11 cells in logarithmic growth phase were treated with different concentrations (100, 200, 300, and 400 nmol/L) of JQ1, 4 nmol/L TPL or different concentrations of JQ1 combined with 4 nmol/L TPL for 48 h. Cell proliferation was detected by CCK-8 method, apoptosis was detected by flow cytometry (FCM), mitochondrial membrane potential was detected by JC-1 method, and expressions of mitochondrial apoptosis pathway-related proteins were detected by Western blot. Results The 50% inhibitory concentration (IC50) value of MV4-11 cells treated with JQ1 for 48 h was (283.9±10.7) nmol/L. However, 4 nmol/L TPL significantly enhanced the inhibitory effect of JQ1 on proliferation of MV4-11 cells, the IC50 value of MV4-11 cells treated with JQ1 combined with TPL was (148.1±2.6) nmol/L, and the difference was statistically significant (t = 25.31, P = 0.029). The result of FCM assay showed that compared with the JQ1 alone group [(9.6±2.3)%, (12.6±1.4)%, (19.5±3.3)%, and (22.7±2.1)%], 4 nmol/L TPL combined with different concentrations (100, 200, 300, and 400 nmol/L) of JQ1 acted on MV4-11 cells for 48 h, the proportions of apoptotic cells were (16.4±1.9)%, (27.5±2.1)%, (32.9±3.6)%, and (35.5±3.0)%, respectively, the difference was statistically significant (F = 9.25, P < 0.01). After treated with 4 nmol/L TPL and JQ1 for 12 h, the level of cell membrane potential in MV4-11 cells was significantly lower than that of JQ1 single agent group, and the difference was statistically significant (P < 0.05). After treated by 4 nmol/L TPL combined with JQ1 for 24 h, the levels of anti-apoptotic proteins bcl-2 and Mcl-1 decreased, and the level of pro-apoptotic protein bax increased. Conclusion TPL can significantly enhance the proliferation inhibition and apoptosis induction effects of BET protein inhibitor JQ1 on MLL-rearranged AML cells, and the mechanism may be related to enhancing the mitochondrial apoptosis pathway. Key words: Gene rearrangement; Leukemia, myeloid, acute; Triptolide; BET protein inhibitor JQ1

IL-23 supports host defense against systemic Candida albicans infection by ensuring myeloid cell survival.

The opportunistic fungal pathogen Candida albicans can cause invasive infections in susceptible hosts and the innate immune system, in particular myeloid cell-mediated immunity, is critical for rapid immune protection and host survival during systemic candidiasis. Using a mouse model of the human disease, we identified a novel role of IL-23 in antifungal defense. IL-23-deficient mice are highly susceptible to systemic infection with C. albicans. We found that this results from a drastic reduction in all subsets of myeloid cells in the infected kidney, which in turn leads to rapid fungal overgrowth and renal tissue injury. The loss in myeloid cells is not due to a defect in emergency myelopoiesis or the recruitment of newly generated cells to the site of infection but, rather, is a consequence of impaired survival of myeloid cells at the site of infection. In fact, the absence of a functional IL-23 pathway causes massive myeloid cell apoptosis upon C. albicans infection. Importantly, IL-23 protects myeloid cells from apoptosis independently of the IL-23-IL-17 immune axis and independently of lymphocytes and innate lymphoid cells. Instead, our results suggest that IL-23 acts in a partially autocrine but not cell-intrinsic manner within the myeloid compartment to promote host protection from systemic candidiasis. Collectively, our data highlight an unprecedented and non-canonical role of IL-23 in securing survival of myeloid cells, which is key for maintaining sufficient numbers of cells at the site of infection to ensure efficient host protection.

MiR-486 promotes proliferation and suppresses apoptosis in myeloid cells by targeting Cebpa in vitro.

The monocytic MDSC (M‐MDSC) is one of the major types of MDSCs, which play important roles in suppression of antitumor immunity. However, the mechanisms underlying how M‐MDSCs so heavily accumulate in patients with cancer are still poorly understood. The purpose of this study was to identify miRNAs that regulate the proliferation and differentiation of M‐MDSCs. Microarray analysis was performed to identify differentially expressed miRNAs between tumor‐induced M‐MDSCs (TM‐MDSCs) and their counterparts from tumor‐free mice. The miRNAs and their target genes that regulate the proliferation and differentiation of myeloid cells were predicted by bioinformatics analysis and validated by RT‐qPCR. Luciferase reporter assays were used to analyze the relationships between miRNAs and target genes. Overexpression of candidate miRNAs and target genes in myeloid cells was conducted to verify their functions in cell proliferation, differentiation, and apoptosis. Our data showed that miR‐486 was overexpressed in TM‐MDSCs. Cebpa was predicted to be one of the target genes of miR‐486 that regulates the proliferation of myeloid cells. Expression of Cebpa was inversely correlated with miR‐486 in TM‐MDSCs, and we found that overexpression of miR‐486 suppressed the expression of Cebpa in both 293T cells determined by luciferase reporter assays and in myeloid cells determined by RT‐qPCR. Overexpression of miR‐486 promoted proliferation and suppressed apoptosis in myeloid cells, as opposed to overexpression of Cebpa, which promoted the opposing phenotype. Overexpression of either miR‐486 or Cebpa inhibited differentiation of myeloid cells. This study indicates that miR‐486 promotes proliferation and suppresses apoptosis in myeloid cells by targeting Cebpa in vitro, suggesting that miR‐486 and Cebpa might be involved in the expansion of TM‐MDSCs in tumor‐bearing mice.

TCEA1 regulates the proliferative potential of mouse myeloid cells

Leukemia is a malignance with complex pathogenesis and poor prognosis. Discovery of noval regulators amenable to leukemia could be of value to gain insight into the pathogenesis, diagnosis and prognosis of leukemia. Here, we conducted a large-scale shRNA library screening for functional regulators in the development of myeloid cells in primary cells. We identified eighteen candidate regulators in the primary screening. Those genes cover a wide range of cellular functions, including gene expression regulation, intracellular signaling transduction, nucleotide excision repair, cell cycle control and transcription regulation. In both primary screening and validation, shRNAs targeting Tcea1, encoding the transcription elongation factor A (SII) 1, exhibited the greatest influence on the proliferative potential of cells. Knocking down the expression of Tcea1 in the 32Dcl3 myeloid cell line led to enhanced proliferation of myeloid cells and blockage of myeloid differentiation induced by G-CSF. In addition, silence of Tcea1 inhibited apoptosis of myeloid cells. Thus, Tcea1 was identified as a gene which can influence the proliferative potential, survival and differentiation of myeloid cells. These findings have implications for how transcriptional elongation influences myeloid cell development and leukemic transformation.

5- Fluorouracil regulation of myeloid-derived suppressor cell differentiation in vitro and in vivo

Abstract The chemotherapeutic agent 5-fluorouracil (5-FU) is the standard therapy for patients with advanced colorectal cancer (CRC). 5-FU not only targets tumor cells for apoptosis but also induces apoptosis in myeloid cells, leading to myelosuppression, which has long been thought as a side effect of 5-FU therapy. Myeloid-derived suppressive cells (MDSCs) are a heterogeneous population of immature myeloid cells that exhibit potent suppressive activity to inhibit T and NK cell function. Recent studies have found that 5-FU suppresses MDSCs in mouse models. However, we observed that MDSCs still massively accumulate in human CRC patients after multiple rounds of 5-FU therapy. We hypothesize that a subset of the heterogeneous MDSCs is resistant to 5-FU and that 5-FU therapy selectively eliminates the sensitive MDSCs, enriching the 5-FU-resistant MDSCs. To test this hypothesis, we made use of both in vitro BM-derived MDSC (BM-MDSC) and in vivo mouse tumor models. We observed that 5-FU therapy significantly decreases CD11b+Gr1+ MDSC accumulation in an orthotopic colon cancer mouse model. BM-MDSCs can be induced by cytokines, including GM-CSF, G-CSF, M-CSF and IL-6, either alone or in combination. Cytological analysis indicates that various cytokines induce BM-MDSC to differentially acquire the morphological appearance of myeloid cell subsets, including macrophage- and granulocyte-like cells. Interestingly, all of these BM-derived MDSCs display resistance to 5-FU-induced apoptosis in vitro following exposure to 5-FU at doses as high as 10 μg/mL. Our data indicates that some MDSC subsets display resistance to 5-FU, providing a rationale to explain the enrichment of MDSCs in human CRC patients following successive treatments with 5-FU.

A Truncated Granulocyte Colony-stimulating Factor Receptor (G-CSFR) Inhibits Apoptosis Induced by Neutrophil Elastase G185R Mutant: IMPLICATION FOR UNDERSTANDING CSF3R GENE MUTATIONS IN SEVERE CONGENITAL NEUTROPENIA

Mutations in ELANE encoding neutrophil elastase (NE) have been identified in the majority of patients with severe congenital neutropenia (SCN). The NE mutants have been shown to activate unfolded protein response and induce premature apoptosis in myeloid cells. Patients with SCN are predisposed to acute myeloid leukemia (AML), and progression from SCN to AML is accompanied by mutations in CSF3R encoding the granulocyte colony-stimulating factor receptor (G-CSFR) in ∼80% of patients. The mutations result in the expression of C-terminally truncated G-CSFRs that promote strong cell proliferation and survival. It is unknown why the CSF3R mutations, which are rare in de novo AML, are so prevalent in SCN/AML. We show here that a G-CSFR mutant, d715, derived from an SCN patient inhibited G-CSF-induced expression of NE in a dominant negative manner. Furthermore, G-CSFR d715 suppressed unfolded protein response and apoptosis induced by an SCN-derived NE mutant, which was associated with sustained activation of AKT and STAT5, and augmented expression of BCL-XL. Thus, the truncated G-CSFRs associated with SCN/AML may protect myeloid precursor cells from apoptosis induced by the NE mutants. We propose that acquisition of CSF3R mutations may represent a mechanism by which myeloid precursor cells carrying the ELANE mutations evade the proapoptotic activity of the NE mutants in SCN patients.

Selective AKR1C3 Inhibitors Potentiate Chemotherapeutic Activity in Multiple Acute Myeloid Leukemia (AML) Cell Lines.

We report the design, synthesis, and evaluation of potent and selective inhibitors of aldo-keto reductase 1C3 (AKR1C3), an important enzyme in the regulatory pathway controlling proliferation, differentiation, and apoptosis in myeloid cells. Combination treatment with the nontoxic AKR1C3 inhibitors and etoposide or daunorubicin in acute myeloid leukemia cell lines, elicits a potent adjuvant effect, potentiating the cytotoxicity of etoposide by up to 6.25-fold and the cytotoxicity of daunorubicin by >10-fold. The results validate AKR1C3 inhibition as a common adjuvant target across multiple AML subtypes. These compounds in coadministration with chemotherapeutics in clinical use enhance therapeutic index and may avail chemotherapy as a treatment option to the pediatric and geriatric population currently unable to tolerate the side effects of cancer drug regimens.

Disruption of Notch signaling aggravates irradiation-induced bone marrow injury, which is ameliorated by a soluble Dll1 ligand through Csf2rb2 upregulation.

Physical and chemical insult-induced bone marrow (BM) damage often leads to lethality resulting from the depletion of hematopoietic stem and progenitor cells (HSPCs) and/or a deteriorated BM stroma. Notch signaling plays an important role in hematopoiesis, but whether it is involved in BM damage remains unclear. In this study, we found that conditional disruption of RBP-J, the transcription factor of canonical Notch signaling, increased irradiation sensitivity in mice. Activation of Notch signaling with the endothelial cell (EC)-targeted soluble Dll1 Notch ligand mD1R promoted BM recovery after irradiation. mD1R treatment resulted in a significant increase in myeloid progenitors and monocytes in the BM, spleen and peripheral blood after irradiation. mD1R also enhanced hematopoiesis in mice treated with cyclophosphamide, a chemotherapeutic drug that induces BM suppression. Mechanistically, mD1R increased the proliferation and reduced the apoptosis of myeloid cells in the BM after irradiation. The β chain cytokine receptor Csf2rb2 was identified as a downstream molecule of Notch signaling in hematopoietic cells. mD1R improved hematopoietic recovery through up-regulation of the hematopoietic expression of Csf2rb2. Our findings reveal the role of Notch signaling in irradiation- and drug-induced BM suppression and establish a new potential therapy of BM- and myelo-suppression induced by radiotherapy and chemotherapy.