Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

IL-3: key orchestrator of inflammation.

Interleukin (IL)-3 has long been known for its hematopoietic properties. However, recent evidence has expanded our understanding of IL-3 function by identifying IL-3 as a critical orchestrator of inflammation in a wide array of diseases. Depending on the type of disease, the course of inflammation, the cell or the tissue involved, IL-3 promotes either pathologic inflammation or its resolution. Here, we describe the cell-specific functions of IL-3 and summarize its role in diseases. We discuss the current treatments targeting IL-3 or its receptor, and highlight the potential and the limitations of targeting IL-3 in clinics.

IL3-Driven T Cell-Basophil Crosstalk Enhances Antitumor Immunity.

Cytotoxic T lymphocytes (CTL) are pivotal in combating cancer, yet their efficacy is often hindered by the immunosuppressive tumor microenvironment, resulting in CTL exhaustion. This study investigates the role of interleukin-3 (IL3) in orchestrating antitumor immunity through CTL modulation. We found that intratumoral CTLs exhibited a progressive decline in IL3 production, which was correlated with impaired cytotoxic function. Augmenting IL3 supplementation, through intraperitoneal administration of recombinant IL3, IL3-expressing tumor cells, or IL3-engineered CD8+ T cells, conferred protection against tumor progression, concomitant with increased CTL activity. CTLs were critical for this therapeutic efficacy as IL3 demonstrated no impact on tumor growth in Rag1 knockout mice or following CD8+ T-cell depletion. Rather than acting directly, CTL-derived IL3 exerted its influence on basophils, concomitantly amplifying antitumor immunity within CTLs. Introducing IL3-activated basophils retarded tumor progression, whereas basophil depletion diminished the effectiveness of IL3 supplementation. Furthermore, IL3 prompted basophils to produce IL4, which subsequently elevated CTL IFNγ production and viability. Further, the importance of basophil-derived IL4 was evident from the absence of benefits of IL3 supplementation in IL4 knockout tumor-bearing mice. Overall, this research has unveiled a role for IL3-mediated CTL-basophil cross-talk in regulating antitumor immunity and suggests harnessing IL3 sustenance as a promising approach for optimizing and enhancing cancer immunotherapy. See related Spotlight, p. 798.

Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain.

Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.

E3 ubiquitin ligase RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα.

IL-3/STAT5 signaling pathway is crucial for the development and activation of immune cells, contributing to the cellular response to infections and inflammatory stimuli. Dysregulation of the IL-3/STAT5 signaling have been associated with inflammatory and autoimmune diseases characterized by inflammatory cell infiltration and organ damage. IL-3 receptor α (IL-3Rα) specifically binds to IL-3 and initiates intracellular signaling, resulting in the phosphorylation of STAT5. However, the regulatory mechanisms of IL-3Rα remain unclear. Here, we identified the E3 ubiquitin ligase RNF128 as a negative regulator of IL-3/STAT5 signaling by targeting IL-3Rα for lysosomal degradation. RNF128 was shown to selectively bind to IL-3Rα, without interacting with the common beta chain IL-3Rß, which shares the subunit with GM-CSF. The deficiency of Rnf128 had no effect on GM-CSF-induced phosphorylation of Stat5, but it resulted in heightened Il-3-triggered activation of Stat5 and increased transcription of the Id1, Pim1, and Cd69 genes. Furthermore, we found that RNF128 promoted the K27-linked polyubiquitination of IL-3Rα in a ligase activity-dependent manner, ultimately facilitating its degradation through the lysosomal pathway. RNF128 inhibited the activation and chemotaxis of macrophages in response to LPS stimulation, thereby attenuating excessive inflammatory responses. Collectively, these results reveal that RNF128 negatively regulates the IL-3/STAT5 signaling pathway by facilitating K27-linked polyubiquitination of IL-3Rα. This study uncovers E3 ubiquitin ligase RNF128 as a novel regulator of the IL-3/STAT5 signaling pathway, providing potential molecular targets for the treatment of inflammatory diseases.

Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.

NFIL3 contributes to cytotoxic T lymphocyte-mediated killing.

Cytotoxic T lymphocytes (CTLs) are key effectors of the adaptive immune system that recognize and eliminate virally infected and cancerous cells. In naive CD8+ T cells, T-cell receptor (TCR) engagement drives a number of transcriptional, translational and proliferation changes over the course of hours and days leading to differentiation into CTLs. To gain a better insight into this mechanism, we compared the transcriptional profiles of naive CD8+ T cells to those of activated CTLs. To find new regulators of CTL function, we performed a selective clustered regularly interspaced short palindromic repeats (CRISPR) screen on upregulated genes and identified nuclear factor IL-3 (NFIL3) as a potential regulator of cytotoxicity. Although NFIL3 has established roles in several immune cells including natural killer, Treg, dendritic and CD4+ T cells, its function in CD8+ CTLs is less well understood. Using CRISPR/Cas9 editing, we found that removing NFIL3 in CTLs resulted in a marked decrease in cytotoxicity. We found that in CTLs lacking NFIL3 TCR-induced extracellular signal-regulated kinase phosphorylation, immune synapse formation and granule release were all intact while cytotoxicity was functionally impaired in vitro. Strikingly, NFIL3 controls the production of cytolytic proteins as well as effector cytokines. Thus, NFIL3 plays a cell intrinsic role in modulating cytolytic mechanisms in CTLs.

Interleukin-3 production by basal-like breast cancer cells is associated with poor prognosis.

Breast cancer represents a collection of pathologies with different molecular subtypes, histopathology, risk factors, clinical behavior, and responses to treatment. "Basal-like" breast cancers predominantly lack the receptors for estrogen and progesterone (ER/PR), lack amplification of human epidermal growth factor receptor 2 (HER2) but account for 10-15% of all breast cancers, are largely insensitive to targeted treatment and represent a disproportionate number of metastatic cases and deaths. Analysis of interleukin (IL)-3 and the IL-3 receptor subunits (IL-3RA + CSF2RB) reveals elevated expression in predominantly the basal-like group. Further analysis suggests that IL-3 itself, but not the IL-3 receptor subunits, associates with poor patient outcome. Histology on patient-derived xenografts supports the notion that breast cancer cells are a significant source of IL-3 that may promote disease progression. Taken together, these observations suggest that IL-3 may be a useful marker in solid tumors, particularly triple negative breast cancer, and warrants further investigation into its contribution to disease pathogenesis.

TRPV1 Channel in Human Eosinophils: Functional Expression and Inflammatory Modulation.

The transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel expressed on sensory neurons and immune cells. We hypothesize that TRPV1 plays a role in human eosinophil function and is modulated by inflammatory conditions. TRPV1 expression on human eosinophils was examined by qPCR, flow cytometry, and immunohistochemistry, respectively. TRPV1 functionality was analyzed by investigating calcium flux, apoptosis, modulation by cytokines and acidic pH, and CD69 externalization using flow cytometry. Activation of TRPV1 induced calcium influx and prolonged survival. Although eosinophils were not directly activated by TRPV1 agonists, activation by IL-3 or GM-CSF was mainly restricted to TRPV1-positive eosinophils. TRPV1 surface content was increased by acidic pH, IL-3, IL-31, IL-33, TSLP, TNF-α, BDNF, and NGF-ß. Interestingly, TRPV1 was also expressed by eosinophils located in proximity to peripheral nerves in atopic dermatitis (AD) skin. In conclusion, eosinophils express functional TRPV1 channels which are increased by extracellular acidification and AD-related cytokines. Since eosinophils also express TRPV1 in AD skin, our results indicate an important role of TRPV1 for neuroimmune interaction mechanisms in itchy, inflammatory skin diseases, like AD.

The transcription factor NFIL3/E4BP4 regulates the developmental stage-specific acquisition of basophil function.

BACKGROUND: Basophils are rare but important effector cells in many allergic disorders. Contrary to their early progenitors, the terminal developmental processes of basophils in which they gain their unique functional properties are unknown. OBJECTIVE: We sought to identify a novel late-stage basophil precursor and a transcription factor regulating the terminal maturation of basophils. METHODS: Using flow cytometry, transcriptome analysis, and functional assays, we investigated the identification and functionality of the basophil precursors as well as basophil development. We generated mice with basophil-specific deletion of nuclear factor IL-3 (NFIL3)/E4BP4 and analyzed the functional impairment of NFIL3/E4BP4-deficient basophils in vitro and in vivo using an oxazolone-induced murine model of allergic dermatitis. RESULTS: We report a new mitotic transitional basophil precursor population (referred to as transitional basophils) that expresses the FcεRIα chain at higher levels than mature basophils. Transitional basophils are less responsive to IgE-linked degranulation but produce more cytokines in response to IL-3, IL-33, or IgE cross-linking than mature basophils. In particular, we found that the expression of NFIL3/E4BP4 gradually rises as cells mature from the basophil progenitor stage. Basophil-specific deletion of NFIL3/E4BP4 reduces the expression of genes necessary for basophil function and impairs IgE receptor signaling, cytokine secretion, and degranulation in the context of murine atopic dermatitis. CONCLUSIONS: We discovered transitional basophils, a novel late-stage mitotic basophil precursor cell population that exists between basophil progenitors and postmitotic mature basophils. We demonstrated that NFIL3/E4BP4 augments the IgE-mediated functions of basophils, pointing to a potential therapeutic regulator for allergic diseases.

Distinct roles of hematopoietic cytokines in the regulation of leukemia stem cells in murine MLL-AF9 leukemia.

Lymphoid-primed multipotent progenitor (LMPP)-like and granulocyte-monocyte progenitor (GMP)-like leukemia stem cells (LSCs) co-exist in the blood of most patients with acute myeloid leukemia (AML). Complete elimination of both types of LSCs is required to cure AML. Using an MLL-AF9-induced murine AML model, we studied the role of hematopoietic cytokines in the survival of LMPP- and GMP-like LSCs. We found that SCF or FLT3L promotes the survival of LMPP-like LSCs by stimulating Stat5-mediated Mcl1 expression, whereas interleukin-3 (IL-3) or IL-6 induces the survival of GMP-like LSCs by stimulating Stat3/nuclear factor κB (NF-κB)-mediated Bcl2 expression. Functional study demonstrated that, compared to AML cells cultured in IL-3 and IL-6 medium, AML cells in SCF- or Flt3L-only culture are highly clonogenic in in vitro culture and are highly leukemogenic in vivo. Our study suggests that co-inhibition of both STAT5-MCL1 and STAT3/NF-κB-BCL2 signaling might represent an improved treatment strategy against AML, specifically AML cases with a monocytic phenotype and/or FLT3 mutations.

NFIL3 aggravates human coronary artery endothelial cell injury by promoting ITGAM transcription in Kawasaki disease.

OBJECTIVE: High expression of nuclear factor interleukin-3 (NFIL3) and integrin Alpha M (ITGAM) was found in serum samples from Kawasaki disease (KD) patients through bioinformatics analysis. Hence, this study aimed to explore the biological functions of NFIL3 and ITGAM in KD serum-stimulated human coronary artery endothelial cells (HCAECs). METHODS: The differentially-expressed genes in KD were analyzed through bioinformatics analysis. Serum samples were obtained from 18 KD patients and 18 healthy volunteers, followed by detection of NFIL3 and ITGAM levels in KD serum. After HCAECs were transfected with sh-NFIL3, sh-ITGAM, or sh-NFIL3 + oe-ITGAM and underwent 24-h KD serum stimulation, cell viability and apoptosis and the levels of inflammation-related factors were measured. The binding between NFIL3 and ITGAM was validated by dual-luciferase and chromatin immunoprecipitation (ChIP) assays. RESULTS: NFIL3 and ITGAM were up-regulated in serum from KD patients and KD serum-stimulated HCAECs. Down-regulation of NFIL3 or ITGAM inhibited KD serum-induced cell apoptosis and inflammatory response of HCAECs and promoted cell viability. Mechanistically, NFIL3 promoted ITGAM transcription level. Up-regulation of ITGAM reversed the improvement of NFIL3 down-regulation on KD serum-induced HCAEC injury. CONCLUSION: NFIL3 aggravated KD serum-induced HCAEC injury by promoting ITGAM transcription, which provided new insights into the treatment of KD.

IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells.

IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r -/- and Il3r +/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.

The Nup98::Nsd1 fusion gene induces CD123 expression in 32D cells.

The NUP98::NSD1 fusion gene is associated with extremely poor prognosis in patients with acute myeloid leukemia (AML). NUP98::NSD1 induces self-renewal and blocks differentiation of hematopoietic stem cells, leading to development of leukemia. Despite its association with poor prognosis, targeted therapy for NUP98::NSD1-positive AML is lacking, as the details of NUP98::NSD1 function are unknown. Here, we generated 32D cells (a murine interleukin-3 (IL-3)-dependent myeloid progenitor cell line) expressing mouse Nup98::Nsd1 to explore the function of NUP98::NSD1 in AML, including comprehensive gene expression analysis. We identified two properties of Nup98::Nsd1 + 32D cells in vitro. First, Nup98::Nsd1 promoted blocking of AML cell differentiation, consistent with a previous report. Second, Nup98::Nsd1 increased dependence on IL-3 for cell proliferation, due to overexpression of the alpha subunit of the IL-3 receptor (IL3-RA, also known as CD123). Consistent with our in vitro data, IL3-RA was also upregulated in samples from patients with NUP98::NSD1-positive AML. These results highlight CD123 as a potential new therapeutic target in NUP98::NSD1-positive AML.

Dark pigmentation and related low FMOD expression increase IL-3 and facilitateplasmacytoid dendritic cell maturation.

According to epidemiological research, skin autoimmune diseases are more prevalent among black Americans. We postulated that pigment-producing melanocytes may contribute to local immune regulation in the microenvironment. We examined murine epidermal melanocytes in vitro to determine the role of pigment production in immune responses mediated by dendritic cell (DC) activation. Our study revealed that darkly pigmented melanocytes produce more IL-3 and the pro-inflammatory cytokines, IL-6 and TNF-α, and consequently induce plasmacytoid DC (pDC) maturation. Additionally, we demonstrate that low pigment-associated fibromodulin (FMOD) interferes with cytokine secretion and subsequent pDC maturation.

JNK signaling during IL-3-mediated differentiation contributes to the c-kit-potentiated allergic inflammatory capacity of mast cells.

Mast cells are leukocytes that mediate various aspects of immunity and drive allergic hypersensitivity pathologies. Mast cells differentiate from hematopoietic progenitor cells in a manner that is largely IL-3 dependent. However, molecular mechanisms, including the signaling pathways that control this process, have yet to be thoroughly investigated. Here, we examine the role of the ubiquitous and critical mitogen-activated protein kinase signaling pathway due to its position downstream of the IL-3 receptor. Hematopoietic progenitor cells were harvested from the bone marrow of C57BL/6 mice and differentiated to bone marrow-derived mast cells in the presence of IL-3 and mitogen-activated protein kinase inhibitors. Inhibition of the JNK node of the mitogen-activated protein kinase pathway induced the most comprehensive changes to the mature mast cell phenotype. Bone marrow-derived mast cells differentiated during impaired JNK signaling expressed impaired c-kit levels on the mast cell surface, first detected at week 3 of differentiation. Following 1 wk of inhibitor withdrawal and subsequent stimulation of IgE-sensitized FcεRI receptors with allergen (TNP-BSA) and c-kit receptors with stem cell factor, JNK-inhibited bone marrow-derived mast cells exhibited impediments in early-phase mediator release through degranulation (80% of control), as well as late-phase secretion of CCL1, CCL2, CCL3, TNF, and IL-6. Experiments with dual stimulation conditions (TNP-BSA + stem cell factor or TNP-BSA alone) showed that impediments in mediator secretion were found to be mechanistically linked to reduced c-kit surface levels. This study is the first to implicate JNK activity in IL-3-mediated mast cell differentiation and also identifies development as a critical and functionally determinative period.

IL-3 produced by T cells is crucial for basophil extravasation in hapten-induced allergic contact dermatitis.

Basophils have been recognized as a characterized cellular player for Th2 immune responses implicated in allergic diseases, but the mechanisms responsible for basophil recruitment to allergic skin remain not well understood. Using a hapten fluorescein isothiocyanate (FITC)-induced allergic contact dermatitis (ACD) mouse model, we show that basophils in FITC-treated IL-3-knockout mice are defective in crossing the vascular endothelium to enter the inflamed skin. By generating mice in which IL-3 is selectively ablated in T cells, we further demonstrate that IL-3 produced by T cells mediates basophil extravasation. Moreover, basophils sorted from FITC-treated IL-3-knockout mice exhibit a decreased expression of integrins Itgam, Itgb2, Itga2b and Itgb7, which are potentially implicated in extravasation process. Interestingly, we observed that these basophils had a reduced expression of retinaldehyde dehydrogenase 1 family member A2 (Aldh1a2), an enzyme responsible for the production of retinoic acid (RA), and administration of all-trans RA restored partially the extravasation of basophils in IL-3-knockout mice. Finally, we validate that IL-3 induces the expression of ALDH1A2 in primary human basophils, and provide further evidence that IL-3 stimulation induces the expression of integrins particularly ITGB7 in an RA-dependent manner. Together, our data propose a model that IL-3 produced by T cells activates ALDH1A2 expression by basophils, leading to the production of RA, which subsequently induces the expression of integrins crucially implicated in basophil extravasation to inflamed ACD skin.

Expression analysis of genes involved in the expansion of hematopoietic stem cells (SCF, Flt3-L, TPO, IL-3, and IL-6) in unrestricted somatic stem cells cultured on fibrin.

Umbilical cord blood (UCB) transplantation is a promising therapeutic approach for patients lacking HLA-matched donors. A main limitation to the use of UCB-derived HSCs (UCB-HSCs) is the low number of transplantable cells. Novel culture strategies are being developed to increase the number of HSCs. Unrestricted somatic stem cells (USSCs) have been identified as promising stromal cells for supporting HSC expansion. The current study aimed to explore the effect of fibrin on the expression of hematopoiesis-related genes (SCF, Flt3-L, TPO, IL-3, and IL-6) in USSCs. USSCs were isolated from UCB and characterized by flow cytometry and in vitro multilineage differentiation ability. DAPI staining and the MTT assay were used to assess the effect of fibrin on USSC viability. The cell attachment was evaluated using SEM. qRT-PCR was performed to evaluate the expression of SCF, Flt3-L, TPO, IL-3, and IL-6 in USSCs cultured on 3D fibrin scaffolds. USSCs were positive for CD73, CD105, and CD166 and negative for CD45. Alizarin red and Oil red O stains confirmed calcium deposition and lipid vacuoles in USSCs. Results obtained from DAPI and MTT assays revealed a positive effect of fibrin on USSC viability. Cells cultured on fibrin express significantly higher levels of SCF and TPO compared to those grown in a 2D environment. The positive effect of fibrin on IL-6 levels was reversed. Fibrin did not affect Flt3-L expression and IL-3 mRNA expression was not detected in either group. The results of this study provide the basis for developing further research on the ex vivo expansion of HSCs with USSCs.

[Effect of wheat-grain moxibustion on Wnt/ß-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition].

OBJECTIVE: To observe the effect of wheat-grain moxibustion at "Dazhui" (GV 14), "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) on Wnt/ß-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 ß-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 ß-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 ß-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/ß-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.