Objective: To investigate the effects of colony-stimulating factor 1 receptor (CSF-1R) inhibitor pexidartinib (PLX3397) on the senescence of bone marrow-derived macrophages (BMDM) stimulated by lipopolysaccharide (LPS). Methods: BMDM were isolated and cultured from femurs and tibiae of 10 male C57BL/6 mice aged 6-8 weeks (obtained from Laboratory Animal Center of Guizhou Medical University). They were divided into blank control group, LPS group (treated with 1 μg/ml LPS for 24 h) as well as low, medium and high concentration PLX3397 pretreatment groups (treated with 100, 500 and 1 000 nmol/L PLX3397 for 4 h respectively followed by 1 μg/ml LPS for 24 h). The corresponding markers of macrophages were detected by flow cytometry. Cell viability was detected by cell counting kit-8 and cellular senescence was detected by senescence-associated-β-galactosidase (SA-β-gal) staining. Meanwhile, protein expressions of cycle-dependent kinase inhibitor p16, p21 and CSF-1R were detected by Western blotting, and the expressions of p16 and p21 were detected by intracellular immunofluorescence. Real-time fluorescence quantitative PCR (RT-qPCR) was used to investigate the mRNA levels of senescence-associated secretory phenotype (SASP) genes including interleukin (IL), IL-1β, chemokine-1/10 (CXCL-1/10), matrix metalloproteinase-8 (MMP-8), and transforming growth factor-β (TGF-β). Results: The rate of SA-β-gal positive staining in medium and high concentration PLX3397 pretreatment groups [(39.33±4.93)% and (36.33±3.06)% respectively] were significantly downregulated compared with LPS group [(52.00±3.00)%] (P=0.020, P=0.005). The expression of CSF-1R protein in low, medium and high concentration PLX3397 pretreatment groups were (0.74±0.18, 0.61±0.07, 0.54±0.06), all of which were significantly lower than that in LPS group (1.16±0.08) (P=0.013, P=0.002, P<0.001). The expression levels of CSF-1R mRNA in low, medium and high concentration PLX3397 pretreatment groups (1.04±0.06, 0.90±0.05, 1.18±0.08) showed similar trend (2.90±0.25) (P<0.001). The average fluorescence intensity of p16 in all PLX3397 pretreatment groups were 49.76±3.65, 48.21±1.72, 47.99±1.26 respectively, which were significantly lower than that in LPS group (66.88±5.85) (P=0.001, P<0.001, P<0.001). The average fluorescence intensity of p21 in medium and high concentration PLX3397 pretreatment groups were (34.43±3.62, 30.13±0.86), significantly lower than that in LPS group (46.82±5.33) (P=0.043, P=0.007). The expression of p16 protein in low, medium and high concentration PLX3397 pretreatment groups (0.56±0.04, 0.55±0.04, 0.35±0.19) were significantly lower than that in LPS group (0.98±0.10) (P=0.003, P=0.002, P<0.001), as well the expression of p21 protein (0.69±0.20, 0.42±0.08, 0.26±0.14) (P=0.032, P=0.002, P<0.001). According to the results of RT-qPCR, the expressions of IL-6, IL-1β, CXCL-1, CXCL-10 and MMP-8 in PLX3397 pretreatment groups were significantly lower than those in LPS group (P<0.001), while the expression of TGF-β increased (P<0.001). Conclusions: LPS could induce the cell senescence, increase the secretion of SASP and aggravate local inflammation by activating the CSF-1R on the cell surface of bone marrow-derived macrophages. CSF-1R inhibitor PLX3397 might attenuate CSF-1R activation associated with LPS and inhibit the senescence of bone marrow-derived macrophages induced by LPS.
Mice ; Animals ; Male ; Lipopolysaccharides/pharmacology* ; Macrophage Colony-Stimulating Factor/metabolism* ; Matrix Metalloproteinase 8/metabolism* ; Mice, Inbred C57BL ; Macrophages ; Transforming Growth Factor beta/metabolism* ; RNA, Messenger/metabolism*

OBJECTIVE: To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition, and to explore the possible mechanism of wheat-grain moxibustion in treating bone marrow inhibition. METHODS: Forty-five SPF male CD1(ICR) mice were randomly divided into a blank group, a model group and a wheat-grain moxibustion group, 15 mice in each group. The bone marrow inhibition model was established by intraperitoneal injection of 80 mg/kg of cyclophosphamide (CTX). The mice in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), 3 moxa cones per acupoint, 30 s per moxa cone, once a day, for 7 consecutive days. The white blood cell count (WBC) was measured before modeling, before intervention and 3, 5 d and 7 d into intervention. After intervention, the general situation of mice was observed; the number of nucleated cells in bone marrow was detected; the serum levels of interleukin-3 (IL-3), interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) were measured by ELISA; the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc in bone marrow cells was measured by Western blot and real-time PCR method. RESULTS: Compared with the blank group, the mice in the model group showed sluggish reaction, unstable gait, decreased body weight, and the WBC, number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were decreased (P<0.01), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was decreased (P<0.01). Compared with the model group, the mice in the wheat-grain moxibustion group showed better general condition, and WBC, the number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were increased (P<0.01, P<0.05), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was increased (P<0.05). CONCLUSION Wheat-grain moxibustion shows therapeutic effect on bone marrow inhibition, and its mechanism may be related to activating Wnt/β-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.
Animals ; Male ; Mice ; beta Catenin/metabolism* ; Bone Marrow/physiopathology* ; Bone Marrow Cells/physiology* ; Granulocyte-Macrophage Colony-Stimulating Factor/metabolism* ; Interleukin-3/metabolism* ; Interleukin-6/metabolism* ; Mice, Inbred ICR ; Moxibustion/methods* ; RNA, Messenger/metabolism* ; Triticum ; Wnt Signaling Pathway ; Hematopoiesis

Aging-induced changes in the immune system are associated with a higher incidence of infection and vaccination failure. Lymph nodes, which filter the lymph to identify and fight infections, play a central role in this process. However, careful characterization of the impact of aging on lymph nodes and associated autoimmune diseases is lacking. We combined single-cell RNA sequencing (scRNA-seq) with flow cytometry to delineate the immune cell atlas of cervical draining lymph nodes (CDLNs) of both young and old mice with or without experimental autoimmune uveitis (EAU). We found extensive and complicated changes in the cellular constituents of CDLNs during aging. When confronted with autoimmune challenges, old mice developed milder EAU compared to young mice. Within this EAU process, we highlighted that the pathogenicity of T helper 17 cells (Th17) was dampened, as shown by reduced GM-CSF secretion in old mice. The mitigated secretion of GM-CSF contributed to alleviation of IL-23 secretion by antigen-presenting cells (APCs) and may, in turn, weaken APCs' effects on facilitating the pathogenicity of Th17 cells. Meanwhile, our study further unveiled that aging downregulated GM-CSF secretion through reducing both the transcript and protein levels of IL-23R in Th17 cells from CDLNs. Overall, aging altered immune cell responses, especially through toning down Th17 cells, counteracting EAU challenge in old mice.
Aging ; Animals ; Autoimmune Diseases ; Disease Models, Animal ; Granulocyte-Macrophage Colony-Stimulating Factor/metabolism* ; Mice ; Mice, Inbred C57BL ; Th17 Cells/metabolism* ; Uveitis/pathology* ; Virulence

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4 helper T (T) cells and CD8 cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of T1 and T2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent T1 or T2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed T1/T2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid T1/T2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.
Antigens ; metabolism ; CTLA-4 Antigen ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Lymphocyte Subsets ; drug effects ; metabolism ; Phenotype ; Proteomics ; Receptors, Chimeric Antigen ; metabolism ; Single-Cell Analysis ; methods ; T-Lymphocytes, Regulatory ; drug effects ; metabolism ; Th1 Cells ; cytology ; drug effects ; Th2 Cells ; cytology ; drug effects ; Transcription, Genetic ; drug effects ; Up-Regulation ; drug effects

Viperin is a multifunctional protein that was first identified in human primary macrophages treated with interferon-γ and in human fibroblasts infected with human cytomegalovirus. This protein plays a role as an anti-viral protein and a regulator of cell signaling pathways or cellular metabolism when induced in a variety of cells such as fibroblasts, hepatocytes and immune cells including T cells and dendritic cells. However, the role of viperin in macrophages is unknown. Here, we show that viperin is basally expressed in murine bone marrow cells including monocytes. Its expression is maintained in bone marrow monocyte-derived macrophages (BMDMs) depending on macrophage colony-stimulating factor (M-CSF) treatment but not on granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. In wild type (WT) and viperin knockout (KO) BMDMs differentiated with M-CSF or G-MCSF, there are little differences at the gene expression levels of M1 and M2 macrophage markers such as inducible nitric oxide synthase (iNOS) and arginase-1, and cytokines such as IL-6 and IL-10, indicating that viperin expression in BMDMs does not affect the basal gene expression of macrophage markers and cytokines. However, when BMDMs are completely polarized, the levels of expression of macrophage markers and secretion of cytokines in viperin KO M1 and M2 macrophages are significantly higher than those in WT M1 and M2 macrophages. The data suggest that viperin plays a role as a regulator in polarization of macrophages and secretion of M1 and M2 cytokines.
Bone Marrow ; Bone Marrow Cells ; Cytokines* ; Cytomegalovirus ; Dendritic Cells ; Fibroblasts ; Gene Expression ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hepatocytes ; Humans ; Interleukin-10 ; Interleukin-6 ; Macrophage Colony-Stimulating Factor ; Macrophages* ; Metabolism ; Monocytes ; Nitric Oxide Synthase Type II ; T-Lymphocytes

OBJECTIVETo explore the formation of pre-metastatic niche in the mouse lung and to study the underlying molecular mechanisms whereby primary breast carcinoma-derived factors mediate recruitment of bone marrow-derived cells (BMDCs) and affect the formation of pre-metastatic lung environment before the arrival of tumor cells.

METHODSMammary carcinoma 4T1 cells were inoculated into the mammary gland to construct mouse model of breast cancer. Confocal microscopy was used to detect the recruitment of BMDCs in the pre-metastatic lungs. The expression of factors in the mouse sera and 4T1 cell culture media was assayed using RayBio Custom mouse cytokine antibody array kit. The mice were injected daily with recombinant VEGF for 7 consecutive days to observe the effect of VEGF on BMDCs recruitment in the mouse lung.

RESULTSNo BMDCs were observed in the lungs of control and 4T1-tumor-bearing mice on day 0. On day 7 and 14, clusters of BMDCs observed in the lungs of 4T1-tumor-bearing mice were 8.7±2.2/objective field and 48.8±3.2/objective field, respectively, significantly higher than those in the control mice (1.1±0.8/objective field and 3.1±1.7/objective field) (P<0.05 for both). Confocal microscopic observation found that metastatic breast cancer cells preferentially facilitate BMDCs recruitment sites in the pre-metastatic mouse lungs. The levels of VEGF, GM-CSF, and IL-6 in the serum of 4T1-tumor-bearing mice were significantly increased compared with those in the control group (P<0.05 for all). However, VEGF was detected only in the culture media of 4T1 cells. The amount of BMDCs in the mouse lung tissue was (22.8±3.6)/objective field in the VEGF group and (3.1±0.4)/objective field in the control group (P<0.05). There were 36.8±5.4 metastatic foci in the lung tissue of VEGF group and 12.6±2.2 in the control group (P<0.05).

CONCLUSIONSThe results of this study demonstrate that primary breast cancer cells can alter the lung microenvironment during the pre-metastatic phase and induce the formation of pre-metastatic niche. Primary tumor cell-derived VEGF may be a crucial factor responsible for the formation of pre-metastatic niche.

Animals ; Bone Marrow Cells ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; blood ; Humans ; Interleukin-6 ; blood ; Lung ; pathology ; Lung Neoplasms ; secondary ; Mice ; Recombinant Proteins ; administration & dosage ; Time Factors ; Tumor Microenvironment ; Vascular Endothelial Growth Factor A ; administration & dosage ; physiology ; secretion

OBJECTIVETo explore the role of epithelial sodium channel (ENaC) in regulating the functional activity of osteoclasts.

METHODSMultinucleated osteoclasts were obtained by inducing the differentiation of rat bone marrow cells with macrophage colony-stimulating factor (M-CSF) and RANKL. The osteoclasts were exposed to different concentrations of the ENaC inhibitor amiloride, and the expression of ENaC on osteoclasts was examined using immunofluorescence technique. The osteoclasts were identified with tartrate-resistant acid phosphatase (TRAP) staining, and the positive cells were incubated with fresh bovine femoral bone slices and the number of bone absorption pits was counted by computer-aided image processing. RT-PCR was performed to analyze the expression of cathepsin K in the osteoclasts.

RESULTSs Exposure to different concentrations of amiloride significantly inhibited the expression of ENaC and reduced the number of TRAP-positive osteoclasts. Exposure of the osteoclasts to amiloride also reduced the number of bone resorption pits on bone slices and the expression of osteoclast-specific gene cathepsin K.

CONCLUSIONs ENaC may participate in the regulation of osteoclast differentiation and bone resorption, suggesting its role in functional regulation of the osteoclasts and a possibly new signaling pathway related with ENaC regulation for modulating bone metabolism.

Animals ; Bone Marrow Cells ; cytology ; Bone Resorption ; Cathepsin K ; metabolism ; Cattle ; Cell Differentiation ; Epithelial Sodium Channels ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Osteoclasts ; cytology ; RANK Ligand ; metabolism ; Rats ; Signal Transduction

OBJECTIVETo investigate the effect of microRNA-382 (miR-382) on the biological properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSC).

METHODSThe mimics and inhibitor of miR-382 were transfected into hUC-MSC with lipo2000. Inverted microscopy was used to observe the morphology change of hUC-MSC. The proliferation of hUC-MSC was detected by CCK-8. Oil red O and alizarin red staining were applied to assess the adipogenic and osteogenic differentiation of hUC-MSC. Cetylpyridinium chloride was used to the quantitative analysis of osteogenic differentiation. The expression of Runx2 and some cytokines were detected by RT-PCR.

RESULTSmiR-382 did not influence the morphology, proliferation and adipogenic differentiation of hUC-MSC miR-382 inhibited the expression of Runx2, thus could inhibit the osteogenesis of hUC-MSC, being confirmed by alizarin red stain; miR-382 could influence the expression of key cytokines secreted from hUC-MSC, such as IL-6, IDO1, G-CSF, M-CSF, GM-CSF.

CONCLUSIONmiR-382 decreases the expression of Runx2 and inhibites the osteogenesis of hUC-MSC. In addition, it also affects the expression of some key cytokines secreted from hUC-MSC.

Cell Differentiation ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Granulocyte Colony-Stimulating Factor ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Interleukin-6 ; metabolism ; Macrophage Colony-Stimulating Factor ; metabolism ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; metabolism ; Osteogenesis ; Transfection ; Umbilical Cord ; cytology

OBJECTIVETo investigate the inducing effect of 'modified' cytokine cocktail on the dendritic cell maturation and migration capability.

METHODSPBMNC were isolated from human peripheral blood stem cell (PBSC) by using density gradient centrifugation, the immature DC (imDC) were induced by using GM-CSF and IL-4 in vitro. Total A549 RNA was transfected into imDC by using electroporation, which was stimulated to matuation by the "gold standard" cytokine cocktail and "modified" cytokine cocktail, respectively. The expression of DC surface markers (CD11c, HLA-DR, CD80, CD83 and CD86) and chemokine receptor (CCR5, CCR7 and CXCR4) were detected by flow cytometry; the mRNA expression levels of DC chemokine receptor (CCR2, CCR5, CCR7, CXCR3 and CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12) were detected by RT-PCR.

RESULTSAs compared with "gold standard cytokine cocktail", the "modified" cytokine cocktail-induced DC expressed higher levels of surface markers (CD11c, HLA-DR, CD80, CD83 and CD86), chemokine receptors (CXCR4) and chemokine (CCL2, CCL3, CCL5, CCL19, CCL21, CXCL10 and CXCL12).

CONCLUSIONThe "modified" cytokine cocktail can more effectively induce the DC maturation, enhace the migratory capability of DC and more generate the immunostimulatory DC, when compared with the "gold standard" cytokine cocktail effect.

Antigens, CD ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Chemokines ; metabolism ; Cytokines ; pharmacology ; Dendritic Cells ; cytology ; drug effects ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Receptors, Chemokine ; metabolism

OBJECTIVETo study the effects of graphene quantum dots (GQDs) on hematopoietic system in rats.

METHODSThirty male SD rats were randomly divided into three groups (n = 10): control group, high dose group (10 mg/kg · d), low dose group (5 mg/kg · d), The rats in experimental group were intravenous injected with GQDs for 28 days and those in control group were injected with normal saline at the same volume. Routine blood and the function of liver and kidney were detected by instrument analysis. The cycle and apoptosis of bone marrow mononuclear cells (BMCs) were detected by FCM. The other three only healthy male SD rat bone marrow mononuclear cells (BMCs) were cultured by joining GQDs for 24 h, 48 h,72 h in vitro, the proliferation was assayed by CCK-8, the content of granulocyte macrophage colony stimulating factor (GM-CSF) from cultural supernatants were detected by ELISA.

RESULTSThe amount of red blood cell and concentration of hemoglobin from experimental group were increased significantly compared with those of control groups (P < 0.05), the concentration of triglyceride and high density lipoprotein were decreased. DNA synthesis period was prolonged (P < 0.01), there was no significant difference in apoptosis. BMCs were promoted proliferation clearly after using GQDs for 72 h (P < 0.05). The content of GM-CSF was increased (P < 0.01) .

CONCLUSIONGQDs may promote hematopoietic function in rats.

Animals ; Apoptosis ; Bone Marrow Cells ; drug effects ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Graphite ; pharmacology ; Hematopoiesis ; drug effects ; Male ; Quantum Dots ; chemistry ; Rats ; Rats, Sprague-Dawley