技术资料

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here,we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1),we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together,our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore,the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures. View Publication

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study,we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae,a nonpathogenic yeast that is rapidly killed and degraded by Mphi,also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin,an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts,whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However,bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus,human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts,whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity. View Publication

Definitive erythropoiesis occurs in islands composed of a central macrophage in contact with differentiating erythroblasts. Erythroid maturation including enucleation can also occur in the absence of macrophages both in vivo and in vitro. We reported previously that loss of Rb induces cell-autonomous defects in red cell maturation under stress conditions,while other reports have suggested that the failure of Rb-null erythroblasts to enucleate is due to defects in associated macrophages. Here we show that erythropoietic islands are disrupted by hypoxic stress,such as occurs in the Rb-null fetal liver,that Rb(-/-) macrophages are competent for erythropoietic island formation in the absence of exogenous stress and that enucleation defects persist in Rb-null erythroblasts irrespective of macrophage function. View Publication

Owing to ongoing recognition of pathogen-associated molecular patterns,immune activation and upregulation of IFN-stimulated genes (ISGs) are sustained in the chronically infected host. Albeit most ISGs are important effectors for containing viral replication,some might exert compensatory immune suppression to limit pathological dysfunctions,although the mechanisms are not fully understood. In this study,we report that the ISG lymphocyte Ag 6 complex,locus E (LY6E) is a negative immune regulator of monocytes. LY6E in monocytes negatively modulated CD14 expression and subsequently dampened the responsiveness to LPS stimulation in vitro. In the setting of chronic HIV infection,the upregulation of LY6E was correlated with reduced CD14 level on monocytes; however,the immunosuppressive effect of LY6E was not adequate to remedy the hyperresponsiveness of activated monocytes. Taken together,the regulatory LY6E pathway in monocytes represents one of negative feedback mechanisms that counterbalance monocyte activation,which might be caused by LPS translocation through the compromised gastrointestinal tract during persistent HIV-1 infection and may serve as a potential target for immune intervention. View Publication

Cytokine responses from monocytes and macrophages exposed to bacteria are of particular importance in innate immunity. Focusing on the impact of the immunoregulatory cytokine interleukin (IL)-27 on control of innate immune system responses,we examined human immune responses to bacterial products and bacterial infection by E. coli and S. typhimurium. Since the effect of IL-27 treatment in human myeloid cells infected with bacteria is understudied,we treated human monocytes and macrophages with IL-27 and either LPS,flagellin,or bacteria,to investigate the effect on inflammatory signaling and cytokine responses. We determined that simultaneous stimulation with IL-27 and LPS derived from E. coli or S. typhimurium resulted in enhanced IL-12p40,TNF-$\alpha$,and IL-6 expression compared to that by LPS alone. To elucidate if IL-27 manipulated the cellular response to infection with bacteria,we infected IL-27 treated human macrophages with S. typhimurium. While IL-27 did not affect susceptibility to S. typhimurium infection or S. typhimurium-induced cell death,IL-27 significantly enhanced proinflammatory cytokine production in infected cells. Taken together,we highlight a role for IL-27 in modulating innate immune responses to bacterial infection. View Publication

IL-27,which is produced by activated APCs,bridges innate and adaptive immunity by regulating the development of Th cells. Recent evidence supports a role for IL-27 in the activation of monocytic cells in terms of inflammatory responses. Indeed,proinflammatory and anti-inflammatory activities are attributed to IL-27,and IL-27 production itself is modulated by inflammatory agents such as LPS. IL-27 primes LPS responses in monocytes; however,the molecular mechanism behind this phenomenon is not understood. In this study,we demonstrate that IL-27 priming results in enhanced LPS-induced IL-6,TNF-α,MIP-1α,and MIP-1β expression in human primary monocytes. To elucidate the molecular mechanisms responsible for IL-27 priming,we measured levels of CD14 and TLR4 required for LPS binding. We determined that IL-27 upregulates TLR4 in a STAT3- and NF-κB-dependent manner. Immunofluorescence microscopy revealed enhanced membrane expression of TLR4 and more distinct colocalization of CD14 and TLR4 upon IL-27 priming. Furthermore,IL-27 priming enhanced LPS-induced activation of NF-κB family members. To our knowledge,this study is the first to show a role for IL-27 in regulating TLR4 expression and function. This work is significant as it reveals new mechanisms by which IL-27 can enhance proinflammatory responses that can occur during bacterial infections. View Publication

The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-lambda1 (interleukin-29 [IL-29]),IFN-lambda2 (IL-28A),and IFN-lambda3 (IL-28B),each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-lambda producers among peripheral lymphocytes. Recently,it has been shown that IFN-lambda1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4(+) T cells and inhibiting the production of Th2-associated cytokines. Here,we asked whether Th2 cytokines exert reciprocal control over IFN-lambda production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-lambda1 transcription and secretion. However,pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments,we showed that IL-4-responsive monocytes are an intermediary cell,responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-lambda1 output. Thus,our experiments revealed a novel mechanism for regulation of both IFN-lambda1 production and pDC function,and suggests an expanded immunomodulatory role for Th2-associated cytokines. View Publication

Interleukin-10 (IL-10) has potent immunoregulatory effects on the maturation and the antigen-presenting cell (APC) function of dendritic cells (DCs). The molecular basis underlying these effects in DCs,however,is ill defined. It is well established that the transcription factor NF-kappaB is a key regulator of DC development,maturation,and APC function. This study was initiated to determine the effects of IL-10 on the NF-kappaB signaling pathway in immature DCs. IL-10 pretreatment of myeloid DCs cultured from bone marrow resulted in reduced DNA binding and nuclear translocation of NF-kappaB after anti-CD40 antibody or lipopolysaccharide (LPS) stimulation. Furthermore,inhibited NF-kappaB activation was characterized by reduced degradation,phosphorylation,or both of IkappaBalpha and IkappaBepsilon but not IkappaBbeta and by reduced phosphorylation of Ser536,located in the trans-activation domain of p65. Notably,IL-10-mediated inhibition of NF-kappaB coincided with suppressed IkappaB kinase (IKK) activity in vitro. Furthermore,IL-10 blocked inducible Akt phosphorylation,and inhibitors of phosphatidylinositol 3-kinase (PI3K) effectively suppressed the activation of Akt,IKK,and NF-kappaB. These findings demonstrate that IL-10 targets IKK activation in immature DCs and that suppressing the PI3K pathway in part mediates blockade of the pathway. View Publication

Although microbial infections can alter steady-state hematopoiesis,the mechanisms that drive such changes are not well understood. We addressed a role for IFN-γ signaling in infection-induced bone marrow suppression and anemia in a murine model of human monocytic ehrlichiosis,an emerging tick-borne disease. Within the bone marrow of Ehrlichia muris-infected C57BL/6 mice,we observed a reduction in myeloid progenitor cells,as defined both phenotypically and functionally. Infected mice exhibited a concomitant increase in developing myeloid cells within the bone marrow,an increase in the frequency of circulating monocytes,and an increase in splenic myeloid cells. The infection-induced changes in progenitor cell phenotype were critically dependent on IFN-γ,but not IFN-α,signaling. In mice deficient in the IFN-γ signaling pathway,we observed an increase in myeloid progenitor cells and CDllb(lo)Gr1(lo) promyelocytic cells within the bone marrow,as well as reduced frequencies of mature granulocytes and monocytes. Furthermore,E. muris-infected IFN-γR-deficient mice did not exhibit anemia or an increase in circulating monocytes,and they succumbed to infection. Gene transcription studies revealed that IFN-γR-deficient CDllb(lo)Gr1(lo) promyelocytes from E. muris-infected mice exhibited significantly reduced expression of irf-1 and irf-8,both key transcription factors that regulate the differentiation of granulocytes and monocytes. Finally,using mixed bone marrow chimeric mice,we show that IFN-γ-dependent infection-induced myelopoiesis occurs via the direct effect of the cytokine on developing myeloid cells. We propose that,in addition to its many other known roles,IFN-γ acts to control infection by directly promoting the differentiation of myeloid cells that contribute to host defense. View Publication

Intestinal fibrosis is a serious complication in inflammatory bowel disease (IBD). Despite the remarkable success of recent anti-inflammatory therapies for IBD,incidence of intestinal fibrosis and need for bowel resection have not significantly changed. To clarify the contribution of haematopoietic-derived cells in intestinal fibrosis,we prepared bone marrow (BM) chimeric mice (chimeras),which were reconstituted with BM cells derived from enhanced green fluorescent protein (EGFP)-transgenic mice or CC chemokine receptor 2 (CCR2)-deficient mice. After 2 months of transplantation,BM chimeras were treated with azoxymethane/dextran sodium sulphate. During chronic inflammation,CCR2+ BM-derived monocyte and fibrocyte infiltration into the colon and CC chemokine ligand 2 production increased,leading to colon fibrosis in EGFP BM chimeras. In CCR2-deficient BM chimeras,monocyte and fibrocyte numbers in the colonic lamina propria significantly decreased,and colon fibrosis was attenuated. In colon tissue,mRNA expression of tissue inhibitor of metalloproteinase (TIMP)-1 but not of collagen I,transforming growth factor-beta1 or matrix metalloproteinases was significantly different between the two chimeras. CCR2+ monocytes and fibrocytes showed high Timp1 mRNA expression. Our results suggest that infiltrating CCR2+ monocytes and their progenies,fibrocytes,promote colon fibrosis by inhibiting collagen degradation through TIMP-1 production. View Publication