技术资料

MSCs have received attention for their therapeutic potential in a number of disease states,including bone formation,diabetes,stem cell engraftment after marrow transplantation,graft-versus-host disease,and heart failure. Despite this diverse interest,the molecular signals regulating MSC trafficking to sites of injury are unclear. MSCs are known to transiently home to the freshly infarcted myocardium. To identify MSC homing factors,we determined chemokine expression pattern as a function of time after myocardial infarction (MI). We merged these profiles with chemokine receptors expressed on MSCs but not cardiac fibroblasts,which do not home after MI. This analysis identified monocyte chemotactic protein-3 (MCP-3) as a potential MSC homing factor. Overexpression of MCP-3 1 month after MI restored MSC homing to the heart. After serial infusions of MSCs,cardiac function improved in MCP-3-expressing hearts (88.7%,p textless .001) but not in control hearts (8.6%,p = .47). MSC engraftment was not associated with differentiation into cardiac myocytes. Rather,MSC engraftment appeared to result in recruitment of myofibroblasts and remodeling of the collagen matrix. These data indicate that MCP-3 is an MSC homing factor; local overexpression of MCP-3 recruits MSCs to sites of injured tissue and improves cardiac remodeling independent of cardiac myocyte regeneration. View Publication

In this study,we have investigated the hypothesis that previously reported beneficial effect of peripheral blood mononuclear cells cultured under angiogenic conditions on cardiovascular function following ischemia is not limited to EPCs but also to monocytes contained therein. We first purified and analyzed the phenotype and secretome of human and murine blood monocytes cultured under angiogenic conditions (named MDs for monocyte derivatives) and tested their effect in a mouse model of myocardial infarction (MI). FACS analysis of MDs shows that these cells express mature endothelial cell markers and that their proliferative capacity is virtually absent,consistent with their end-differentiated monocytic ontogeny. MDs secreted significant levels of HGF,IGF-1,MCP-1,and sTNFR-1 relative to their monocyte precursors. MDs were unable to form vascular networks in vitro when cultured on matrix coated flasks. Treatment of murine HL-1 cardiomyocyte cell line with MD-conditioned medium reduced their death induced by TNF-alpha,staurosporine,and oxidative stress,and this effect was dependent upon MD-derived sTNFR-1,HGF,and IGF-1. We further demonstrate that MD secretome promoted endothelial cell proliferation and capacity to form vessels in vitro and this was dependent upon MD-derived MCP-1,HGF,and IGF-1. Echocardiography analysis showed that MD myocardial implantation improved left ventricle fractional shortening of mouse hearts following MI and was associated with reduced myocardial fibrosis and enhancement of angiogenesis. Transplanted MDs and their secretome participate in preserving functional myocardium after ischemic insult and attenuate pathological remodeling. View Publication

A variety of nonmalignant cells present in the tumor microenvironment promotes tumorigenesis by stimulating tumor cell growth and metastasis or suppressing host immunity. The role of such stromal cells in T-cell lymphoproliferative disorders is incompletely understood. Monocyte-derived cells (MDCs),including professional antigen-presenting cells such as dendritic cells (DCs),play a central role in T-cell biology. Here,we provide evidence that monocytes promote the survival of malignant T cells and demonstrate that MDCs are abundant within the tumor microenvironment of T cell-derived lymphomas. Malignant T cells were observed to remain viable during in vitro culture with autologous monocytes,but cell death was significantly increased after monocyte depletion. Furthermore,monocytes prevent the induction of cell death in T-cell lymphoma lines in response to either serum starvation or doxorubicin,and promote the engraftment of these cells in nonobese diabetic/severe combined immunodeficient mice. Monocytes are actively recruited to the tumor microenvironment by CCL5 (RANTES),where their differentiation into mature DCs is impaired by tumor-derived interleukin-10. Collectively,the data presented demonstrate a previously undescribed role for monocytes in T-cell lymphoproliferative disorders. View Publication

Genital herpes,caused by herpes simplex virus type 2 (HSV-2),is one of the most prevalent sexually transmitted diseases worldwide and a risk factor for acquiring human immunodeficiency virus. Although many vaccine candidates have shown promising results in animal models,they have failed to be effective in human trials. In this study,a humanized mouse strain was evaluated as a potential preclinical model for studying human immune responses to HSV-2 infection and vaccination. Immunodeficient mouse strains were examined for their abilities to develop human innate and adaptive immune cells after transplantation of human umbilical cord stem cells. A RAG2(-/-) gammac(-/-) mouse strain with a BALB/c background was chosen as the most appropriate model and was then examined for its ability to mount innate and adaptive immune responses to intravaginal HSV-2 infection and immunization. After primary infection,human cells in the lymph nodes were able to generate a protective innate immune response and produce gamma interferon (IFN-gamma). After intravaginal immunization and infection,human T cells and NK cells were found in the genital tract and iliac lymph nodes. In addition,human T cells in the spleen,lymph nodes,and vaginal tract were able to respond to stimulation with HSV-2 antigens by replicating and producing IFN-gamma. Human B cells were also able to produce HSV-2-specific immunoglobulin G. These adaptive responses were also shown to be protective and reduce local viral replication in the genital tract. This approach provides a means for studying human immune responses in vivo using a small-animal model and may become an important preclinical tool. View Publication

The lipocalin mouse 24p3 has been implicated in diverse physiological processes,including apoptosis,iron trafficking,development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However,a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3,we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid,myeloid,and erythroid cells,which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead,apoptotic defects were unique to many mature hematopoietic cell types,including neutrophils,cytokine-dependent mast cells,thymocytes,and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells,thus explaining their resistance to the ensuing cell death. The results of these studies,in conjunction with those of previous studies,reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly,these functions are limited to relatively mature blood cell compartments. View Publication

The differentiation of effector CD8(+) T cells is a dynamically regulated process that varies during different infections and is influenced by the inflammatory milieu of the host. In this study,we define three signals regulating CD8(+) T cell responses during tuberculosis by focusing on cytokines known to affect disease outcome: IL-12,type I IFN,and IL-27. Using mixed bone marrow chimeras,we compared wild-type and cytokine receptor knockout CD8(+) T cells within the same mouse following aerosol infection with Mycobacterium tuberculosis. Four weeks postinfection,IL-12,type 1 IFN,and IL-27 were all required for efficient CD8(+) T cell expansion in the lungs. We next determined if these cytokines directly promote CD8(+) T cell priming or are required only for expansion in the lungs. Using retrogenic CD8(+) T cells specific for the M. tuberculosis Ag TB10.4 (EsxH),we observed that IL-12 is the dominant cytokine driving both CD8(+) T cell priming in the lymph node and expansion in the lungs; however,type I IFN and IL-27 have nonredundant roles supporting pulmonary CD8(+) T cell expansion. Thus,IL-12 is a major signal promoting priming in the lymph node,but a multitude of inflammatory signals converge in the lung to promote continued expansion. Furthermore,these cytokines regulate the differentiation and function of CD8(+) T cells during tuberculosis. These data demonstrate distinct and overlapping roles for each of the cytokines examined and underscore the complexity of CD8(+) T cell regulation during tuberculosis. View Publication

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid,massively parallel profiling of transcriptomes. However,assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool,demuxlet,that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data,we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples,demuxlet correctly recovers the sample identity of<99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples. View Publication

Like their human counterparts,mouse plasmacytoid dendritic cells (pDCs) play a central role in innate immunity against viral infections,but their capacity to prime T cells in vivo remains unknown. We show here that virus-activated pDCs differentiate into antigen-presenting cells able to induce effector/memory CD8(+) T-cell responses in vivo against both epitopic peptides and endogenous antigen,whereas pDCs activated by synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG) acquire only the ability to recall antigen-experienced T-cell responses. We also show that immature pDCs are unable to induce effector or regulatory CD8(+) T-cell responses. Thus,murine pDCs take part in both innate and adaptive immune responses by directly priming naive CD8(+) T cells during viral infection. View Publication

Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live,but not killed,virulent MTB. Additionally,we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live,virulent MTB,and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases,and by knockdown of crucial transcriptional regulators,we demonstrate that protein kinase-A (PKA),CREB,and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis,we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live,virulent MTB to subvert the immune system to survive and replicate in the host. View Publication

Human and mouse studies indicate that TLRs are important in mycobacterial infections. We investigated TLR gene expression in fresh unstimulated blood and bronchoalveolar lavage from patients with pulmonary tuberculosis using a well-validated,real-time PCR. A human splice variant of TLR1,designated hsTLR1,was found in all donors tested. hsTLR1 mRNA lacks exon 2,which is a 77-bp region of the 5'-untranslated region,but contains the same coding sequence as TLR1. Compared with the matched controls,whole blood from patients had increased levels of mRNA encoding TLR2 (p = 0.0006),TLR1 (p = 0.004),hsTLR1 (p = 0.0003),TLR6 (p textless 0.0001),and TLR4 (p = 0.0002). By contrast,expression of these TLRs was not increased in bronchoalveolar lavage. An increased level of hsTLR1 mRNA was found in both CD3- (p = 0.0078) and CD4+ cells (p = 0.028),resulting in an increased ratio of hsTLR1 mRNA to TLR1 and to TLR6 mRNA. An in vitro study in THP1 cells suggested that this relative increase in hsTLR1 might be attributable to a direct effect of mycobacterial components because it could be mimicked by mycobacterial preparations in the absence of IFN-gamma or T cells and by the TLR1/2 agonist Pam3CysK4. Half-life studies using blood from patients with pulmonary tuberculosis and THP1 cells exposed to Myobacterium tuberculosis in vitro showed p38 MAPK-independent stabilization of mRNAs encoding hsTLR1 and TLR1. We conclude that M. tuberculosis exerts direct effects on patterns of TLR expression,partly via changes in mRNA half-life. The significance of these changes in the pathogenesis of disease deserves further investigation. View Publication