技术资料

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However,little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb),97A6,that specifically detects human Ba,MC (lung,skin),and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils,eosinophils,monocytes,lymphocytes,platelets) did not react with MoAb 97A6,and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (textgreater98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40),Ba-eosinophil colonies (7 of 40),Ba-macrophage colonies (3 of 40),and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast,no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells,starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (textgreater30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors,MC progenitors,Ba progenitors,as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors. View Publication

Leukocyte recruitment to inflammation sites progresses in a multistep cascade. Chemokines regulate multiple steps of the cascade,including arrest,transmigration,and chemotaxis. The most important chemokine receptor in mouse neutrophils is CXCR2,which couples through G$\alpha$i2- and G$\alpha$i3-containing heterotrimeric G proteins. Neutrophils arrest in response to CXCR2 stimulation. This is defective in G$\alpha$i2-deficient neutrophils. In this study,we show that G$\alpha$i3-deficient neutrophils showed reduced transmigration but normal arrest in mice. We also tested G$\alpha$i2- or G$\alpha$i3-deficient neutrophils in a CXCL1 gradient generated by a microfluidic device. G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils showed significantly reduced migration and directionality. This was confirmed in a model of sterile inflammation in vivo. G$\alpha$i2-,but not G$\alpha$i3-,deficient neutrophils showed decreased Ca(2+) flux in response to CXCR2 stimulation. Conversely,G$\alpha$i3-,but not G$\alpha$i2-,deficient neutrophils exhibited reduced AKT phosphorylation upon CXCR2 stimulation. We conclude that G$\alpha$i2 controls arrest and G$\alpha$i3 controls transmigration and chemotaxis in response to chemokine stimulation of neutrophils. View Publication

Maintaining a steady pool of self-renewing hematopoietic stem cells (HSCs) is critical for sustained production of multiple blood lineages. Many transcription factors and molecules involved in chromatin and epigenetic modifications have been found to be critical for HSC self-renewal and differentiation; however,their interplay is less understood. The transcription factor GA binding protein (GABP),consisting of DNA-binding subunit GABPα and transactivating subunit GABPβ,is essential for lymphopoiesis as shown in our previous studies. Here we demonstrate cell-intrinsic,absolute dependence on GABPα for maintenance and differentiation of hematopoietic stem/progenitor cells. Through genome-wide mapping of GABPα binding and transcriptomic analysis of GABPα-deficient HSCs,we identified Zfx and Etv6 transcription factors and prosurvival Bcl-2 family members including Bcl-2,Bcl-X(L),and Mcl-1 as direct GABP target genes,underlying its pivotal role in HSC survival. GABP also directly regulates Foxo3 and Pten and hence sustains HSC quiescence. Furthermore,GABP activates transcription of DNA methyltransferases and histone acetylases including p300,contributing to regulation of HSC self-renewal and differentiation. These systematic analyses revealed a GABP-controlled gene regulatory module that programs multiple aspects of HSC biology. Our studies thus constitute a critical first step in decoding how transcription factors are orchestrated to regulate maintenance and multipotency of HSCs. View Publication

BACKGROUND: Critical limb ischemia due to peripheral arterial occlusive disease is associated with a severely increased morbidity and mortality. There is no effective pharmacological therapy available. Injection of autologous bone marrow-derived mononuclear cells (BM-MNC) is a promising therapeutic option in patients with critical limb ischemia,but double-blind,randomized trials are lacking. METHODS AND RESULTS: Forty patients with critical limb ischemia were included in a multicenter,phase II,double-blind,randomized-start trial to receive either intraarterial administration of BM-MNC or placebo followed by active treatment with BM-MNC (open label) after 3 months. Intraarterial administration of BM-MNC did not significantly increase ankle-brachial index and,thus,the trial missed its primary end point. However,cell therapy was associated with significantly improved ulcer healing (ulcer area,3.2±4.7 cm(2) to 1.89±3.5 cm(2) [P=0.014] versus placebo,2.92±3.5 cm(2) to 2.89±4.1 cm(2) [P=0.5]) and reduced rest pain (5.2±1.8 to 2.2±1.3 [P=0.009] versus placebo,4.5±2.4 to 3.9±2.6 [P=0.3]) within 3 months. Limb salvage and amputation-free survival rates did not differ between the groups. Repeated BM-MNC administration and higher BM-MNC numbers and functionality were the only independent predictors of improved ulcer healing. Ulcer healing induced by repeated BM-MNC administration significantly correlated with limb salvage (r=0.8; Ptextless0.001). CONCLUSIONS: Intraarterial administration of BM-MNC is safe and feasible and accelerates wound healing in patients without extensive gangrene and impending amputation. These exploratory findings of this pilot trial need to be confirmed in a larger randomized trial in patients with critical limb ischemia and stable ulcers. View Publication

SerpinB1 is among the most efficient inhibitors of neutrophil serine proteases--NE,CG,and PR-3--and we investigated here its role in neutrophil development and homeostasis. We found that serpinB1 is expressed in all human bone marrow leukocytes,including stem and progenitor cells. Expression levels were highest in the neutrophil lineage and peaked at the promyelocyte stage,coincident with the production and packaging of the target proteases. Neutrophil numbers were decreased substantially in the bone marrow of serpinB1(-/-) mice. This cellular deficit was associated with an increase in serum G-CSF levels. On induction of acute pulmonary injury,neutrophils were recruited to the lungs,causing the bone marrow reserve pool to be completely exhausted in serpinB1(-/-) mice. Numbers of myeloid progenitors were normal in serpinB1(-/-) bone marrow,coincident with the absence of target protease expression at these developmental stages. Maturation arrest of serpinB1(-/-) neutrophils was excluded by the normal CFU-G growth in vitro and the normal expression in mature neutrophils of early and late differentiation markers. Normal absolute numbers of proliferating neutrophils and pulse-chase kinetic studies in vivo showed that the bone marrow deficit in serpinB1(-/-) mice was largely restricted to mature,postmitotic neutrophils. Finally,upon overnight culture,apoptosis and necrosis were greater in purified bone marrow neutrophils from serpinB1(-/-) compared with WT mice. Collectively,these findings demonstrate that serpinB1 sustains a healthy neutrophil reserve that is required in acute immune responses. View Publication

We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD),a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production,reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs,with subsequent selection and full characterization to ensure no off-target changes,holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs." View Publication

We recently demonstrated that lack of type I IFN signaling (IFNAR knockout) in lymphocyte-deficient mice (IFrag(-/-)) results in bone marrow (BM) failure after Pneumocystis lung infection,whereas lymphocyte-deficient mice with intact IFNAR (RAG(-/-)) had normal hematopoiesis. In the current work,we performed studies to define further the mechanisms involved in the induction of BM failure in this system. BM chimera experiments revealed that IFNAR expression was required on BM-derived but not stroma-derived cells to prevent BM failure. Signals elicited after day 7 postinfection appeared critical in determining BM cell fate. We observed caspase-8- and caspase-9-mediated apoptotic cell death,beginning with neutrophils. Death of myeloid precursors was associated with secondary oxidative stress,and decreasing colony-forming activity in BM cell cultures. Treatment with N-acetylcysteine could slow the progression of,but not prevent,BM failure. Type I IFN signaling has previously been shown to expand the neutrophil life span and regulate the expression of some antiapoptotic factors. Quantitative RT-PCR demonstrated reduced mRNA abundance for the antiapoptotic factors BCL-2,IAP2,MCL-1,and others in BM cells from IFrag(-/-) compared with that in BM cells from RAG(-/-) mice at day 7. mRNA and protein for the proapoptotic cytokine TNF-α was increased,whereas mRNA for the growth factors G-CSF and GM-CSF was reduced. In vivo anti-TNF-α treatment improved precursor cell survival and activity in culture. Thus,we propose that lack of type I IFN signaling results in decreased resistance to inflammation-induced proapoptotic stressors and impaired replenishment by precursors after systemic responses to Pneumocystis lung infection. Our finding may have implications in understanding mechanisms underlying regenerative BM depression/failure during complex immune deficiencies such as AIDS. View Publication

Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation,however,neutrophils are necessary for optimal viral control. In this study,we explore the role of the nucleotide-binding domain leucine-rich repeat containing receptor family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp12-/- mice are protected from lethality during IAV infection and show decreased vascular permeability,fewer pulmonary neutrophils,and a reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared with wild-type mice. Nlrp12-/- neutrophils and dendritic cells within the IAV-infected lungs produce less CXCL1 than their wild-type counterparts. Decreased CXCL1 production by Nlrp12-/- dendritic cells was not due to a difference in CXCL1 protein stability,but instead to a decrease in Cxcl1 mRNA stability. Together,these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1-mediated neutrophilic responses. View Publication

MicroRNAs (miRNAs) are small,noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-,maturation-,and disease-specific miRNA expression has been described,miRNA-dependent phenotypes and miRNA-regulated signaling in hematopoietic cells are largely unknown. Combining functional genomics,biochemical analysis,and unbiased and hypothesis-driven miRNA target prediction,we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells,miR-125b over-expression enhances colony-formation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression,respectively. However,gene-specific RNAi reveals that this reduction,alone and in combination,is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression,DNA-binding,and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression,indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed,we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/miR-125b cells. Interestingly,gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. View Publication

Eosinophils contribute to type II immune responses in helminth infections and allergic diseases; however,their influence on intracellular pathogens is less clear. We previously reported that CCR2(-/-) mice exposed to the intracellular fungal pathogen Histoplasma capsulatum exhibit dampened immunity caused by an early exaggerated interleukin (IL)-4 response. We sought to identify the cellular source promulgating IL-4 in infected mutant animals. Eosinophils were the principal instigators of non-protective IL-4 and depleting this granulocyte population improved fungal clearance in CCR2(-/-) animals. The deleterious impact of eosinophilia on mycosis was also recapitulated in transgenic animals overexpressing eosinophils. Mechanistic examination of IL-4 induction revealed that phagocytosis of H. capsulatum via the pattern recognition receptor complement receptor (CR) 3 triggered the heightened IL-4 response in murine eosinophils. This phenomenon was conserved in human eosinophils; exposure of cells to the fungal pathogen elicited a robust IL-4 response. Thus,our findings elucidate a detrimental attribute of eosinophil biology in fungal infections that could potentially trigger a collapse in host defenses by instigating type II immunity.Mucosal Immunology advance online publication,6 April 2016; doi:10.1038/mi.2016.26. View Publication

Mobile sensing based on the integration of microfluidic device and smartphone,so-called MS2 technology,has enabled many applications over recent years,and continues to stimulate growing interest in both research communities and industries. In particular,it has been envisioned that MS2 technology can be developed for various cell functional assays to enable basic research and clinical applications. Toward this direction,in this paper,we describe the development of a MS2-based cell functional assay for testing cell migration (the Mkit). The system is constructed as an integrated test kit,which includes microfluidic chips,a smartphone-based imaging platform,the phone apps for image capturing and data analysis,and a set of reagent and accessories for performing the cell migration assay. We demonstrated that the Mkit can effectively measure purified neutrophil and cancer cell chemotaxis. Furthermore,neutrophil chemotaxis can be tested from a drop of whole blood using the Mkit with red blood cell (RBC) lysis. The effects of chemoattractant dose and gradient profile on neutrophil chemotaxis were also tested using the Mkit. In addition to research applications,we demonstrated the effective use of the Mkit for on-site test at the hospital and for testing clinical samples from chronic obstructive pulmonary disease patient. Thus,this developed Mkit provides an easy and integrated experimental platform for cell migration related research and potential medical diagnostic applications. View Publication

IL-17A is a T cell-derived proinflammatory cytokine required for microbial host defense. In vivo expression profoundly stimulates granulopoiesis. At baseline,the hemopoietic system of IL-17R knockout mice (IL-17Ra(-/-)) is,with the exception of increased splenic progenitor numbers,indistinguishable from normal control mice. However,when challenged with gamma irradiation,hemopoietic toxicity is significantly more pronounced in IL-17Ra(-/-) animals,with the gamma irradiation-associated LD(50) being reduced by 150 rad. In spleen-derived T cells,gamma irradiation induces significant murine IL-17A expression in vivo but not in vitro. After sublethal radiation injury (500 rad),the infusion of purified CD4(+) T cells enhances hemopoietic recovery. This recovery is significantly impaired in IL-17Ra(-/-) animals or after in vivo blockade of IL-17Ra in normal mice,resulting in a reduction of hemopoietic precursors by 50% and of neutrophils by 43%. Following sublethal radiation-induced myelosuppression,in vivo overexpression of murine IL-17A in normal mice substantially enhanced granulopoietic restoration in mice with a 4-fold increase in neutrophils and splenic precursors on day 8 (CFU-granulocyte-macrophage/granulocyte-erythrocyte-megakaryocyte-monocyte,CFU-high proliferative potential),as well as 2- and 3-fold increases of bone marrow precursors,respectively. This establishes IL-17A as a hemopoietic response cytokine to radiation injury in mice and an inducible mechanism that is required for recovery of granulopoiesis after radiation injury. View Publication