技术资料

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation,reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization,and the cytoplasmic domain propagates signals that activate β(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly� View Publication

The present study shows that alumina nanotopography affects monocyte/macrophage behavior. Human mononuclear cells cultured on alumina membranes with pore diameters of 20 and 200 nm were evaluated in terms of cell adhesion,viability,morphology,and release of proinflammatory cytokines. After 24 hours,cell adhesion was assessed by means of light microscopy and cell viability by measuring LDH release. The inflammatory response was evaluated by quantifying interleukin-1β and tumour necrosis factor-α. Finally,scanning electron microscopy was used to study cell morphology. Results showed pronounced differences in cell number,morphology,and cytokine release depending on the nanoporosity. Few but highly activated cells were found on the 200 nm porous alumina,while relatively larger number of cells were found on the 20 nm porous surface. However,despite their larger number,the cells adhering on the 20 nm surface exhibited reduced pro-inflammatory activity. The data of this paper implies that nanotopography could be exploited for controlling the inflammatory response to implants. View Publication

Polymorphonuclear neutrophils (PMNs) are critical for the formation,maintenance,and resolution of bacterial abscesses. However,the mechanisms that regulate PMN survival and proliferation during the evolution of an abscess are not well defined. Using a mouse model of Staphylococcus aureus abscess formation within a cutaneous wound,combined with real-time imaging of genetically tagged PMNs,we observed that a high bacterial burden elicited a sustained mobilization of PMNs from the bone marrow to the infected wound,where their lifespan was markedly extended. A continuous rise in wound PMN number,which was not accounted for by trafficking from the bone marrow or by prolonged survival,was correlated with the homing of c-kit(+)-progenitor cells from the blood to the wound,where they proliferated and formed mature PMNs. Furthermore,by blocking their recruitment with an antibody to c-kit,which severely limited the proliferation of mature PMNs in the wound and shortened mouse survival,we confirmed that progenitor cells are not only important contributors to PMN expansion in the wound,but are also functionally important for immune protection. We conclude that the abscess environment provides a niche capable of regulating PMN survival and local proliferation of bone marrow-derived c-kit(+)-progenitor cells. View Publication

Granulocyte colony-stimulating factor (G-CSF),the prototypical mobilizing cytokine,induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated,in part,through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization,osteoblast suppression,and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact,demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover,G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally,we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together,these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance,ultimately leading to HSPC mobilization. View Publication

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies,the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case,the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity. View Publication

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

Tumor-associated macrophages (TAMs) originate as circulating monocytes,and are recruited to gliomas,where they facilitate tumor growth and migration. Understanding the interaction between TAM and cancer cells may identify therapeutic targets for glioblastoma multiforme (GBM). Vascular cell adhesion molecule-1 (VCAM-1) is a cytokine-induced adhesion molecule expressed on the surface of cancer cells,which is involved in interactions with immune cells. Analysis of the glioma patient database and tissue immunohistochemistry showed that VCAM-1 expression correlated with the clinico-pathological grade of gliomas. Here,we found that VCAM-1 expression correlated positively with monocyte adhesion to GBM,and knockdown of VCAM-1 abolished the enhancement of monocyte adhesion. Importantly,upregulation of VCAM-1 is dependent on epidermal-growth-factor-receptor (EGFR) expression,and inhibition of EGFR effectively reduced VCAM-1 expression and monocyte adhesion activity. Moreover,GBM possessing higher EGFR levels (U251 cells) had higher VCAM-1 levels compared to GBMs with lower levels of EGFR (GL261 cells). Using two- and three-dimensional cultures,we found that monocyte adhesion to GBM occurs via integrin α4β1,which promotes tumor growth and invasion activity. Increased proliferation and tumor necrosis factor-α and IFN-γ levels were also observed in the adherent monocytes. Using a genetic modification approach,we demonstrated that VCAM-1 expression and monocyte adhesion were regulated by the miR-181 family,and lower levels of miR-181b correlated with high-grade glioma patients. Our results also demonstrated that miR-181b/protein phosphatase 2A-modulated SP-1 de-phosphorylation,which mediated the EGFR-dependent VCAM-1 expression and monocyte adhesion to GBM. We also found that the EGFR-dependent VCAM-1 expression is mediated by the p38/STAT3 signaling pathway. Our study suggested that VCAM-1 is a critical modulator of EGFR-dependent interaction of monocytes with GBM,which raises the possibility of developing effective and improved therapies for GBM.Oncogene advance online publication,1 May 2017; doi:10.1038/onc.2017.129. View Publication

Autologous chondrocyte implantation is the current gold standard cell therapy for cartilage lesions. However,in some instances,the heavily compromised health of the patient can either impair or limit the recovery of the autologous chondrocytes and a satisfactory outcome of the implant. Allogeneic human articular chondrocytes (hAC) could be a good alternative,but the possible immunological incompatibility between recipient and hAC donor should be considered. Herein,we report that allogeneic hAC inhibited T lymphocyte response to antigen-dependent and -independent proliferative stimuli. This effect was maximal when T cells and hAC were in contact and it was not relieved by the addition of exogenous lymphocyte growth factor interleukin (IL)-2. More important,hAC impaired the differentiation of peripheral blood monocytes induced with granulocyte monocyte colony-stimulating factor and IL-4 (Mo) to professional antigen-presenting cells,such as dendritic cells (DC). Indeed,a marked inhibition of the onset of the CD1a expression and an ineffective downregulation of CD14 antigens was observed in Mo-hAC co-cultures. Furthermore,compared to immature or mature DC,Mo from Mo-hAC co-cultures did not trigger an efficacious allo-response. The prostaglandin (PG) E2 present in the Mo-hAC co-culture conditioned media is a putative candidate of the hAC-mediated inhibition of Mo maturation. Altogether,these findings indicate that allogeneic hAC inhibit,rather than trigger,immune response and strongly suggest that an efficient chondrocyte implantation could be possible also in an allogeneic setting. View Publication

Virulent intracellular pathogens,such as the Salmonella species,engage numerous virulence factors to subvert host defence mechanisms to induce a chronic infection that leads to typhoid or exacerbation of other chronic inflammatory conditions. Here we show the role of the forkhead transcription factor FoxO3a during infection of mice with Salmonella typhimurium (ST). Although FoxO3a signalling does not affect the development of CD8(+) T cell responses to ST,FoxO3a has an important protective role,particularly during the chronic stage of infection,by limiting the persistence of oxidative stress. Furthermore,FoxO3a signalling regulates ERK signalling in macrophages,which results in the maintenance of a proinflammatory state. FoxO3a signalling does not affect cell proliferation or cell death. Thus,these results reveal mechanisms by which FoxO3a promotes host survival during infection with chronic,virulent intracellular bacteria. View Publication

IL-18 is a member of the IL-1 family involved in innate immunity and inflammation. Deregulated levels of IL-18 are involved in the pathogenesis of multiple disorders including inflammatory and metabolic diseases,yet relatively little is known regarding its regulation. Liver X receptors or LXRs are key modulators of macrophage cholesterol homeostasis and immune responses. Here we show that LXR ligands negatively regulate LPS-induced mRNA and protein expression of IL-18 in bone marrow-derived macrophages. Consistent with this being an LXR-mediated process,inhibition is abolished in the presence of a specific LXR antagonist and in LXR-deficient macrophages. Additionally,IL-18 processing of its precursor inactive form to its bioactive state is inhibited by LXR through negative regulation of both pro-caspase 1 expression and activation. Finally,LXR ligands further modulate IL-18 levels by inducing the expression of IL-18BP,a potent endogenous inhibitor of IL-18. This regulation occurs via the transcription factor IRF8,thus identifying IL-18BP as a novel LXR and IRF8 target gene. In conclusion,LXR activation inhibits IL-18 production through regulation of its transcription and maturation into an active pro-inflammatory cytokine. This novel regulation of IL-18 by LXR could be applied to modulate the severity of IL-18 driven metabolic and inflammatory disorders. View Publication

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development,and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver,whereby perturbations of one tissue can influence behavior of the other. Here,we present a study on human gut-liver tissue interactions under normal and inflammatory contexts,via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells),and intestinal (enterocytes,goblet cells,and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g.,barrier integrity) and hepatic (e.g.,albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut,versus isolation,revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover,significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example,production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk,with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g.,liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648-2659. textcopyright 2017 Wiley Periodicals,Inc. View Publication

Macrophages are a source of both proinflammatory and restorative functions in damaged tissue through complex dynamic phenotypic changes. Here,we sought to determine whether monocyte-derived macrophages (MDMs) contribute to recovery after acute sterile brain injury. By profiling the transcriptional dynamics of MDMs in the murine brain after experimental intracerebral hemorrhage (ICH),we found robust phenotypic changes in the infiltrating MDMs over time and demonstrated that MDMs are essential for optimal hematoma clearance and neurological recovery. Next,we identified the mechanism by which the engulfment of erythrocytes with exposed phosphatidylserine directly modulated the phenotype of both murine and human MDMs. In mice,loss of receptor tyrosine kinases AXL and MERTK reduced efferocytosis of eryptotic erythrocytes and hematoma clearance,worsened neurological recovery,exacerbated iron deposition,and decreased alternative activation of macrophages after ICH. Patients with higher circulating soluble AXL had poor 1-year outcomes after ICH onset,suggesting that therapeutically augmenting efferocytosis may improve functional outcomes by both reducing tissue injury and promoting the development of reparative macrophage responses. Thus,our results identify the efferocytosis of eryptotic erythrocytes through AXL/MERTK as a critical mechanism modulating macrophage phenotype and contributing to recovery from ICH. View Publication