The nuclear factor ID3 endows macrophages with a potent anti-tumour activity.

Macrophage activation is controlled by a balance between activating and inhibitory receptors1-7, which protect normal tissues from excessive damage during infection8,9 but promote tumour growth and metastasis in cancer7,10. Here we report that the Kupffer cell lineage-determining factor ID3 controls this balance and selectively endows Kupffer cells with the ability to phagocytose live tumour cells and orchestrate the recruitment, proliferation and activation of natural killer and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumours. ID3 shifts the macrophage inhibitory/activating receptor balance to promote the phagocytic and lymphoid response, at least in part by buffering the binding of the transcription factors ELK1 and E2A at the SIRPA locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumour activity to mouse bone-marrow-derived macrophages and human induced pluripotent stem-cell-derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumour niche, which could be harnessed for cell therapy in cancer.

Bone marrow plasma cells require P2RX4 to sense extracellular ATP.

Plasma cells produce large quantities of antibodies and so play essential roles in immune protection1. Plasma cells, including a long-lived subset, reside in the bone marrow where they depend on poorly defined microenvironment-linked survival signals1. We show that bone marrow plasma cells use the ligand-gated purinergic ion channel P2RX4 to sense extracellular ATP released by bone marrow osteoblasts through the gap-junction protein pannexin 3 (PANX3). Mutation of Panx3 or P2rx4 each caused decreased serum antibodies and selective loss of bone marrow plasma cells. Compared to their wild-type counterparts, PANX3-null osteoblasts secreted less extracellular ATP and failed to support plasma cells in vitro. The P2RX4-specific inhibitor 5-BDBD abrogated the impact of extracellular ATP on bone marrow plasma cells in vitro, depleted bone marrow plasma cells in vivo and reduced pre-induced antigen-specific serum antibody titre with little posttreatment rebound. P2RX4 blockade also reduced autoantibody titre and kidney disease in two mouse models of humoral autoimmunity. P2RX4 promotes plasma cell survival by regulating endoplasmic reticulum homeostasis, as short-term P2RX4 blockade caused accumulation of endoplasmic reticulum stress-associated regulatory proteins including ATF4 and B-lineage mutation of the pro-apoptotic ATF4 target Chop prevented bone marrow plasma cell demise on P2RX4 inhibition. Thus, generating mature protective and pathogenic plasma cells requires P2RX4 signalling controlled by PANX3-regulated extracellular ATP release from bone marrow niche cells.

CTRP6 promotes the macrophage inflammatory response, and its deficiency attenuates LPS-induced inflammation.

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.

The Effect of CaV1.2 Inhibitor Nifedipine on Chondrogenic Differentiation of Human Bone Marrow or Menstrual Blood-Derived Mesenchymal Stem Cells and Chondrocytes.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa2+) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa2+ was highest in chondrocytes, whereas iCa2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa2+, Cav1.2 and the proliferation of all cells and affected iCa2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

Hydroxysafflor Yellow A-Induced Osteoblast Differentiation and Proliferation of BM-MSCs by Up-Regulating Nuclear Vitamin D Receptor.

BACKGROUND: Vitamin D receptor (VDR) is critical for mineral and bone homeostasis since it plays an essential role in the osteoblast differentiation of bone marrow mesenchymal stem cells (BM-MSCs). Hydroxysafflor yellow A (HSYA) has the potential to promote bone mineralization and inhibit bone resorption, while its detailed mechanism needs to be elaborated. OBJECTIVE: This study intends to explore the action of HSYA on the proliferation and differentiation of BM-MSC and the underlying mechanism. METHODS: Different concentrations of HSYA to BM-MSC and CCK-8, and EdU were used to detect cell viability and proliferation. The alkaline phosphatase (ALP) was used to observe the differentiation ability of BM-MSC osteoblasts. The calcium uptake and mineralization of osteoblast-like cells were observed by alizarin red staining. The level of calcium ion uptake in cells was detected by flow cytometry. AutoDock was performed for molecular docking of HSYA to VDR protein. Immunofluorescence and western blotting were performed to detect the expression of VDR expression levels. Finally, the effect of VDR was verified by a VDR inhibitor. RESULTS: After treatment with HSYA, the proliferation and calcium uptake of BM-MSC were increased. The level of ALP increased significantly and reached its peak on the 12th day. HSYA promoted calcium uptake and calcium deposition, and mineralization of osteoblasts. The western blotting and immunofluorescence showed that HSYA increased the expression of VDR in the osteoblast-like cell's nucleus and upregulated Osteocalcin, S100 calcium-binding protein G, and CYP24A1. In addition, HYSA treatment increased the expression of osteopontin and the synthesis of osteogenic proteins, such as Type 1 collagen. After the addition of the VDR inhibitor, the effect of HSYA was weakened. CONCLUSION: HSYA could significantly promote the activity and proliferation of osteoblasts and increase the expression level of VDR in osteoblasts. HSYA may also improve calcium absorption by osteoblasts by regulating the synthesis of calciumbinding protein and vitamin D metabolic pathway-related proteins.

Bone marrow mesenchymal stem cells facilitate diabetic wound healing through the restoration of epidermal cell autophagy via the HIF-1α/TGF-ß1/SMAD pathway.

BACKGROUND: The biological activity and regenerative medicine of bone marrow mesenchymal stem cells (BMSCs) have been focal topics in the broad fields of diabetic wound repair. However, the molecular mechanisms are still largely elusive for other cellular processes that are regulated during BMSC treatment. Our previous studies have shown that hypoxia is not only a typical pathological phenomenon of wounds but also exerts a vital regulatory effect on cellular bioactivity. In this study, the beneficial effects of hypoxic BMSCs on the cellular behaviors of epidermal cells and diabetic wound healing were investigated. METHOD: The viability and secretion ability of hypoxic BMSCs were detected. The autophagy, proliferation and migration of HaCaT cells cultured with hypoxic BMSCs-derived conditioned medium were assessed by estimating the expression of autophagy-related proteins, MTS, EdU proliferation and scratch assays. And the role of the SMAD signaling pathway during hypoxic BMSC-evoked HaCaT cell autophagy was explored through a series of in vitro gain- and loss-of-function experiments. Finally, the therapeutic effects of hypoxic BMSCs were evaluated using full-thickness cutaneous diabetic wound model. RESULTS: First, we demonstrated that hypoxic conditions intensify HIF-1α-mediated TGF-ß1 secretion by BMSCs. Then, the further data revealed that BMSC-derived TGF-ß1 was responsible for the activation of epidermal cell autophagy, which contributed to the induction of epidermal cell proliferation and migration. Here, the SMAD signaling pathway was identified as downstream of BMSC-derived TGF-ß1 to regulate HaCaT cell autophagy. Moreover, the administration of BMSCs to diabetic wounds increased epidermal autophagy and the rate of re-epithelialization, leading to accelerated healing, and these effects were significantly attenuated, accompanied by the downregulation of Smad2 phosphorylation levels due to TGF-ß1 interference in BMSCs. CONCLUSION: In this report, we present evidence that uncovers a previously unidentified role of hypoxic BMSCs in regulating epidermal cell autophagy. The findings demonstrate that BMSC-based treatment by restoring epidermal cell autophagy could be an attractive therapeutic strategy for diabetic wounds and that the process is mediated by the HIF-1α/TGF-ß1/SMAD pathway.

IL-1ß expression in bone marrow dendritic cells is induced by TLR2 agonists and regulates HSC function.

Hematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1ß (IL-1ß) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1-/- mice show that TLR1/2-induced HSPC expansion is dependent on IL-1ß signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1ß and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1ß expression.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Effect of miR-381-3p/FGF7 axis on the osteogenic differentiation of bone marrow mesenchymal stem cells through MEK/ERK signaling pathway.

Although microRNAs (miRNAs) exert an important role in the osteogenesis of mesenchymal stem cells (MSCs), the effect of miR-381-3p on the osteogenic differentiation in MBD­MSCs is still unclear. The BMMSCs from patients with MBD (MBD­MSC) or normal participants (Normal­MSC) were isolated and induced to differentiation with dexamethasone. BMMSCs were transfected with miR-381-3p mimic, miR-381-3p inhibitor, and FGF7 siRNA to regulate the expression of miR-381-3p or FGF7. The direct binding between miR-381-3p and FGF7 was predicted and confirmed by bioinformatics prediction and luciferase reporter assay. The effect of miR-381-3p on the osteogenic differentiation of BMMSCs was assessed by RT­qPCR, alizarin Red S staining and western blot assays. Isolated BMMSCs showed the regular morphology, and were positive for CD44, CD90 and CD105 but negative for CD34 and CD45 markers. The calcium deposition and the relative mRNA expression levels of ALP, OC and OPN after induction were markedly enhanced. MiR-381-3p was upregulated in BMMSCs. Also, inhibition of miR-381-3p notably promoted osteogenic differentiation, vice versa. Besides, miR-381-3p could directly target FGF7 and negatively modulate the expression of FGF7. Moreover, inhibition of FGF7 attenuated the increase of the calcium deposition, and the relative mRNA expression of ALP, OC and OPN caused by the downregulation of miR-381-3p. In addition, the miR-381-3p inhibitor-induced the enhancement of the relative protein expressions of FGFR2, p-MEK and p-ERK1/2 were significantly reduced by the co-transfection of si-FGF7. Furthermore, the application of LY3214996, the inhibitor of ERK also verified these outcomes. MiR-381-3p directly targeting FGF7 modulated the osteogenic differentiation via MEK/ERK signaling pathway in BMMSCs.

Bmal1- and Per2-mediated regulation of the osteogenic differentiation and proliferation of mouse bone marrow mesenchymal stem cells by modulating the Wnt/ß-catenin pathway.

BACKGROUND: Bmal1 and Per2 are the core components of the circadian clock genes (CCGs). Bmal1-/- mice exhibit premature aging, as indicated by hypotrichosis and osteoporosis, with a loss of proliferation ability. The same occurs in Per2-/- mice, albeit to a less severe degree. However, whether the effects of Bmal1 and Per2 on proliferation and osteogenic differentiation are synergistic or antagonistic remains unclear. Thus, our study aimed to explore the effects and specific mechanism. METHODS AND RESULTS: Lentiviral and adenoviral vectors were constructed to silence or overexpress Bmal1 or Per2 and MTT, flow cytometry, RT-qPCR, WB, immunohistochemistry, alizarin red staining and ChIP-Seq analyses were applied to identify the possible mechanism. The successful knockdown and overexpression of Bmal1/Per2 were detected by fluorescence microcopy. Flow cytometry found out that Bmal1 or Per2 knockdown resulted in G1-phase cell cycle arrest. RT-qPCR showed the different expression levels of Wnt-3a, c-myc1 and axin2 in the Wnt/ß-catenin signaling pathway as well as the gene expression change of Rorα and Rev-erbα. Meanwhile, related proteins such as ß-catenin, TCF-1, and P-GSK-3ß were detected. ALP activity and the amount of mineral nodules were compared. ChIP-Seq results showed the possible mechanism. CONCLUSIONS: Bmal1 and Per2, as primary canonical clock genes, showed synergistic effects on the proliferation and differentiation of BMSCs. They would inhibit the Wnt/ß-catenin signaling pathway by downregulating Rorα expression or upregulating Rev-erbα expression, both of which were also key elements of CCGs. And this may be the mechanism by which they negatively regulate the osteogenic differentiation of BMSCs. Bmal1 and Per2 show synergistic effects in the proliferation of BMSCs. In addition, they play a synergistic role in negatively regulating the osteogenic differentiation ability of BMSCs. Bmal1 and Per2 may regulate the aging of BMSCs by altering cell proliferation and osteogenic differentiation through Rorα and Rev-erbα to affect Wnt/ß-catenin pathway.

A Novel Xeno-Free Method to Isolate Human Endometrial Mesenchymal Stromal Cells (E-MSCs) in Good Manufacturing Practice (GMP) Conditions.

The cyclic regeneration of human endometrium is guaranteed by the proliferative capacity of endometrial mesenchymal stromal cells (E-MSCs). Due to this, the autologous infusion of E-MSCs has been proposed to support endometrial growth in a wide range of gynecological diseases. We aimed to compare two different endometrial sampling methods, surgical curettage and vacuum aspiration biopsy random assay (VABRA), and to validate a novel xeno-free method to culture human E-MSCs. Six E-MSCs cell samples were isolated after mechanical tissue homogenization and cultured using human platelet lysate. E-MSCs were characterized for the colony formation capacity, proliferative potential, and multilineage differentiation. The expression of mesenchymal and stemness markers were tested by FACS analysis and real-time PCR, respectively. Chromosomal alterations were evaluated by karyotype analysis, whereas tumorigenic capacity and invasiveness were tested by soft agar assay. Both endometrial sampling techniques allowed efficient isolation and expansion of E-MSCs using a xeno-free method, preserving their mesenchymal and stemness phenotype, proliferative potential, and limited multi-lineage differentiation ability during the culture. No chromosomal alterations and invasive/tumorigenic capacity were observed. Herein, we report the first evidence of efficient E-MSCs isolation and culture in Good Manufacturing Practice compliance conditions, suggesting VABRA endometrial sampling as alternative to surgical curettage.

UM171A-induced ROS promote antigen cross-presentation of immunogenic peptides by bone marrow-derived mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been extensively used in the clinic due to their exquisite tissue repair capacity. However, they also hold promise in the field of cellular vaccination as they can behave as conditional antigen presenting cells in response to interferon (IFN)-gamma treatment under a specific treatment regimen. This suggests that the immune function of MSCs can be pharmacologically modulated. Given the capacity of the agonist pyrimido-indole derivative UM171a to trigger the expression of various antigen presentation-related genes in human hematopoietic progenitor cells, we explored the potential use of UM171a as a means to pharmacologically instill and/or promote antigen presentation by MSCs. METHODS: Besides completing a series of flow-cytometry-based phenotypic analyses, several functional antigen presentation assays were conducted using the SIINFEKL-specific T-cell clone B3Z. Anti-oxidants and electron transport chain inhibitors were also used to decipher UM171a's mode of action in MSCs. Finally, the potency of UM171a-treated MSCs was evaluated in the context of therapeutic vaccination using immunocompetent C57BL/6 mice with pre-established syngeneic EG.7T-cell lymphoma. RESULTS: Treatment of MSCs with UM171a triggered potent increase in H2-Kb cell surface levels along with the acquisition of antigen cross-presentation abilities. Mechanistically, such effects occurred in response to UM171a-mediated production of mitochondrial-derived reactive oxygen species as their neutralization using anti-oxidants or Antimycin-A mitigated MSCs' ability to cross-present antigens. Processing and presentation of the immunogenic ovalbumin-derived SIINFEKL peptide was caused by de novo expression of the Psmb8 gene in response to UM171a-triggered oxidative stress. When evaluated for their anti-tumoral properties in the context of therapeutic vaccination, UM171a-treated MSC administration to immunocompetent mice with pre-established T-cell lymphoma controlled tumor growth resulting in 40% survival without the need of additional supportive therapy and/or standard-of-care. CONCLUSIONS: Altogether, our findings reveal a new immune-related function for UM171a and clearly allude to a direct link between UM171a-mediated ROS induction and antigen cross-presentation by MSCs. The fact that UM171a treatment modulates MSCs to become antigen-presenting cells without the use of IFN-gamma opens-up a new line of investigation to search for additional agents capable of converting immune-suppressive MSCs to a cellular tool easily adaptable to vaccination.

Alkbh1-mediated DNA N6-methyladenine modification regulates bone marrow mesenchymal stem cell fate during skeletal aging.

OBJECTIVES: DNA N6-methyladenine (N6-mA) demethylase Alkbh1 participates in regulating osteogenic differentiation of mesenchymal stem cell (MSCs) and vascular calcification. However, the role of Alkbh1 in bone metabolism remains unclear. MATERIALS AND METHODS: Bone marrow mesenchymal stem cells (BMSCs)-specific Alkbh1 knockout mice were used to investigate the role of Alkbh1 in bone metabolism. Western blot, qRT-PCR, and immunofluorescent staining were used to evaluate the expression of Alkbh1 or optineurin (optn). Micro-CT, histomorphometric analysis, and calcein double-labeling assay were used to evaluate bone phenotypes. Cell staining and qRT-PCR were used to evaluate the osteogenic or adipogenic differentiation of BMSCs. Dot blotting was used to detect the level of N6-mA in genomic DNA. Chromatin immunoprecipitation (Chip) assays were used to identify critical targets of Alkbh1. Alkbh1 adeno-associated virus was used to overexpress Alkbh1 in aged mice. RESULTS: Alkbh1 expression in BMSCs declined during aging. Knockout of Alkbh1 promoted adipogenic differentiation of BMSCs while inhibited osteogenic differentiation. BMSC-specific Alkbh1 knockout mice exhibited reduced bone mass and increased marrow adiposity. Mechanistically, we identified optn as the downstream target through which Alkbh1-mediated DNA m6A modification regulated BMSCs fate. Overexpression of Alkbh1 attenuated bone loss and marrow fat accumulation in aged mice. CONCLUSIONS: Our findings demonstrated that Alkbh1 regulated BMSCs fate and bone-fat balance during skeletal aging and provided a potential target for the treatment of osteoporosis.

Clinical utility of the CD10+/HLA-DR+ population in bone marrow mononuclear cells from adults with immune thrombocytopenia.

Several patients with immune thrombocytopenia show good clinical courses without any major complications. However, severe bleeding complications, such as hemoptysis, gastrointestinal bleeding, and intracranial hemorrhages, are occasionally observed in some patients associated with marked thrombocytopenia; this results in 1.5-fold higher mortality for such patients compared with the general population. We report here the cases of two patients with immune thrombocytopenia whose bone marrow included a prominent cluster of differentiation (CD)10+/ human leukocyte antigen (HLA)-DR+ population and showed good response to steroid therapy. Conversely, two other patients without a CD10+/HLA-DR+ population were refractory to steroids, and one of them had a serious course. Retrospective examination of 30 patients with severe immune thrombocytopenia revealed that they had a higher percentage of CD10+/HLA-DR+ cells compared with patients with other benign hematological diseases. As differential diagnosis of immune thrombocytopenia and aplastic anemia with severe thrombocytopenia is often difficult, it may be helpful to understand whether CD10+/HLA-DR+ cells are increased. We also show the possible correlation of resistance to steroid therapy and lower percentages of CD10+/HLA-DR+ cells. It has been reported that nonresponsiveness to steroid treatment was a high risk factor for intracranial hemorrhage. Lower percentages of CD10+/HLA-DR+ cells may be a useful tool to identify patients with immune thrombocytopenia at a high risk of serious bleeding complications.

Siglec-F Promotes IL-33-Induced Cytokine Release from Bone Marrow-Derived Eosinophils Independently of the ITIM and ITIM-like Motif Phosphorylation.

Eosinophils are potent innate effector cells associated mainly with type 2 immune responses elicited by helminths and allergens. Their activity needs to be tightly controlled to prevent severe inflammation and tissue damage. Eosinophil degranulation and secretion of inflammatory effector molecules, including cytokines, chemokines, and lipid mediators, can be regulated by activating and inhibitory receptors on the cell surface. In this study, we investigated the modulation of proliferation, apoptosis, gene expression, and cytokine/chemokine secretion from IL-33-activated Mus musculus eosinophils on cross-linking of the transmembrane receptor Sialic acid-binding Ig-like lectin F (Siglec-F). Siglec-F contains an ITIM plus an ITIM-like motif in its intracellular tail and is mainly regarded as an inhibitory and apoptosis-inducing receptor. In vitro costimulation of bone marrow-derived eosinophils with anti-Siglec-F and IL-33 compared with treatment with either alone led to enhanced STAT6 phosphorylation, stronger induction of hypoxia/glycolysis-related proinflammatory genes, and elevated secretion of type 2 cytokines (IL-4, IL-13) and chemokines (CCL3, CCL4) with only minor effects on proliferation and apoptosis. Using a competitive mixed bone marrow chimera approach with wild-type and Siglec-F-deficient eosinophils, we observed no evidence for Siglec-F-regulated inhibition of Aspergillus fumigatus-elicited lung eosinophilia. Truncation of the Siglec-F cytoplasmic tail, but not mutation of the ITIM and ITIM-like motifs, ablated the effect of enhanced cytokine/chemokine secretion. This provides evidence for an ITIM phosphorylation-independent signaling pathway from the cytoplasmic tail of the Siglec-F receptor that enhances effector molecule release from activated eosinophils.

Two birds with one stone: YQSSF regulates both proliferation and apoptosis of bone marrow cells to relieve chemotherapy-induced myelosuppression.

ETHNOPHARMACOLOGICAL RELEVANCE: Yiqi Shengsui formula (YQSSF) is a commonly used formula to treat chemotherapy-induced myelosuppression, but little is known about its therapeutic mechanisms. AIM OF THIS STUDY: This study aims to examine the effect of YQSSF in treating myelosuppression and explore its mechanism. MATERIALS AND METHODS: A myelosuppression BALB/c mouse model was established by intraperitoneal (i.p.) injection of cyclophosphamide (CTX). The efficacy of YQSSF in alleviating chemotherapy-induced myelosuppression was evaluated by blood cell count, immune organ (thymus, spleen, liver) index, bone marrow nucleated cell (BMNC) count and histopathological analysis of bone marrow and spleen. Then, ultra-performance liquid chromatograph quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to analyze the ingredients of YQSSF extract. Key effects and potential mechanism of YQSSF extract in alleviating myelosuppression were predicted by network pharmacology method. Finally, cell cycle and TUNEL staining of bone marrow cells was detected to verify the key effects, and RT-qPCR or Western blotting were performed to measure the gene and protein expressions of the effect targets respectively to confirm the predicted mechanism of YQSSF for myelosuppression. RESULTS: YQSSF up-regulated the number of peripheral blood leukocytes and BMNC, reduced spleen index and liver index, improved the pathological morphology of bone marrow and spleen. A total of 40 ingredients were isolated from YQSSF extract using UPLC-Q/TOF-MS analysis. Network pharmacology revealed that YQSSF regulated both proliferation and apoptosis to alleviate myelosuppression. Finally, YQSSF decreased G0/G1 ratio, increased the proportion of bone marrow cells in S phase and proliferation index (PI), and reduced apoptotic cells in femur bone marrow. RT-qPCR and Western blotting showed that YQSSF up-regulated the expression levels of CDK4, CDK6, CyclinB1, c-Myc and Bcl-2, as well as down-regulated the expression levels of Cyt-c, Fas, Caspase-8/3 and p53. CONCLUSIONS: YQSSF promotes the proliferation and inhibits the apoptosis of bone marrow cells to relieve chemotherapy-induced myelosuppression.

Heme-stress activated NRF2 skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages in hemolytic anemia.

Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias, such as sickle cell disease. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Since chronic heme-stress is noxious for macrophages, erythrophagocytes in the spleen are continuously replenished from bone marrow-derived progenitors. Here, we hypothesized that adaptation to heme stress progressively shifts differentiation trajectories of bone marrow progenitors to expand the capacity of heme-handling monocyte-derived macrophages at the expense of the homeostatic generation of dendritic cells, which emerge from shared myeloid precursors. This heme-induced redirection of differentiation trajectories may contribute to hemolysis-induced secondary immunodeficiency. We performed single-cell RNA-sequencing with directional RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation of bone marrow cells towards antioxidant, iron-recycling macrophages, suppressing the generation of dendritic cells in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific CD4 T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype of macrophage expansion with concurrent dendritic cell depletion was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning of hemolytic stress as a driver of hyposplenism-related secondary immunodeficiency.

Structural Characterization of Polysaccharides Isolated from Panax notoginseng Medicinal Residue and Its Protective Effect on Myelosuppression Induced by Cyclophosphamide.

This study aims to establish the isolation and purification method of polysaccharides from medicinal residue of Panax notoginseng (PPN). The structure and protective effect of PPN on myelosuppression mice were investigated. One neutral polysaccharide (NPPN) and five acidic polysaccharides (APPN I, APPN II-A, APPN II-B, APPN III-A, and APPN III-B) were obtained. The results confirmed that NPPN, APPN I and APPN II-A are glycan with 1, 4 main chains. APPN III-A is a glycan. APPN II-B and APPN III-B are homogalacturonan pectin with 1, 4 main chains. This study demonstrated that NPPN played a bone marrow protective role in myelosuppression mice induced by cyclophosphamide. NPPN could relieve cell cycle arrest, reduce the apoptosis rate of marrow cells, and improve granulocyte-macrophage colony-stimulating (GM-CSF), thermoplastic polyolefin (TPO) and erythropoietin (EPO) serum level, which contributes to promoting the proliferation of hematopoietic cells.

Heterogeneous expression of ACE2 and TMPRRS2 in mesenchymal stromal cells.

The outbreak of COVID-19 has become a serious public health emergency. The virus targets cells by binding the ACE2 receptor. After infection, the virus triggers in some humans an immune storm containing the release of proinflammatory cytokines and chemokines followed by multiple organ failure. Several vaccines are enrolled, but an effective treatment is still missing. Mesenchymal stem cells (MSCs) have shown to secrete immunomodulatory factors that suppress this cytokine storm. Therefore, MSCs have been suggested as a potential treatment option for COVID-19. We report here that the ACE2 expression is minimal or nonexistent in MSC derived from three different human tissue sources (adipose tissue, umbilical cord Wharton`s jelly and bone marrow). In contrast, TMPRSS2 that is implicated in SARS-CoV-2 entry has been detected in all MSC samples. These results are of particular importance for future MSC-based cell therapies to treat severe cases after COVID-19 infection.

More than neutrophils: Lin(+)Ly6G(+)IL-5Rα(+) multipotent myeloid cells (MMCs) are dominant in normal murine bone marrow and retain capacity to differentiate into eosinophils and monocytes.

Bone marrow is a hematopoietic site harboring multiple populations of myeloid cells in different stages of differentiation. Murine bone marrow eosinophils are traditionally identified by Siglec-F(+) staining using flow cytometry, whereas neutrophils are characterized by Ly6G(+) expression. However, using flow cytometry to characterize bone marrow hematopoietic cells in wild-type mice, we found substantial gray areas in identification of these cells. Siglec-F(+) mature eosinophil population constituted only a minority of bone marrow Lin(+)CD45(+) pool (5%). A substantial population of Siglec-F(-) cells was double positive for neutrophil marker Ly6G and eosinophil lineage marker, IL-5Rα. This granulocyte population with mixed neutrophil and eosinophil characteristics is typically attributable to neutrophil pool based on neutral granule staining and expression of Ly6G and myeloid peroxidase. It is distinct from Lineage(-) myeloid progenitors or Siglec-F(+)Ly6G(+) maturing eosinophil precursors, and can be accurately identified by Lineage(+) staining and positive expression of markers IL-5Rα and Ly6G. At 15-50% of all CD45(+) hematopoietic cells in adult mice (percentage varies by sex and age), this is a surprisingly dominant population, which increases with age in both male and female mice. RNA-seq characterization of these cells revealed a complex immune profile and the capacity to secrete constituents of the extracellular matrix. When sorted from bone marrow, these resident cells had neutrophilic phenotype but readily acquired all characteristics of eosinophils when cultured with G-CSF or IL-5, including expression of Siglec-F and granular proteins (Epx, Mbp). Surprisingly, these cells were also able to differentiate into Ly6C(+) monocytes when cultured with M-CSF. Herein described is the discovery of an unexpected hematopoietic flexibility of a dominant population of multipotent myeloid cells, typically categorized as neutrophils, but with the previously unknown plasticity to contribute to mature pools of eosinophils and monocytes.