技术资料

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here,we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model,HMBA did not reactivate HIV-1,yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However,HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells,hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside,HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall,our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells,providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug. View Publication

Lentiviral vectors are the method of choice for stable gene modification of a variety of cell types. However,the efficiency with which they transduce target cells varies significantly,in particular their typically poor capacity to transduce primary stem cells. Here we describe the isolation and enrichment of murine bone-marrow mesenchymal stem cells (MSCs) via fluorescence-activated cell sorting (FACS); the cloning,production,and concentration of high-titer second generation lentiviral vectors via combined tangential flow filtration (TFF) and ultracentrifugation; and the subsequent high-efficiency gene modification of MSCs into insulin-producing cells via overexpression of the furin-cleavable human insulin (INS-FUR) gene. View Publication

Human immunodeficiency virus type 1 (HIV-1) eradication is prevented by the establishment on infection of cellular HIV-1 reservoirs that are not fully characterized,especially in genital mucosal tissues (the main HIV-1 entry portal on sexual transmission). Here,we show,using penile tissues from HIV-1-infected individuals under suppressive combination antiretroviral therapy,that urethral macrophages contain integrated HIV-1 DNA,RNA,proteins and intact virions in virus-containing compartment-like structures,whereas viral components remain undetectable in urethral T cells. Moreover,urethral cells specifically release replication-competent infectious HIV-1 following reactivation with the macrophage activator lipopolysaccharide,while the T-cell activator phytohaemagglutinin is ineffective. HIV-1 urethral reservoirs localize preferentially in a subset of polarized macrophages that highly expresses the interleukin-1 receptor,CD206 and interleukin-4 receptor,but not CD163. To our knowledge,these results are the first evidence that human urethral tissue macrophages constitute a principal HIV-1 reservoir. Such findings are determinant for therapeutic strategies aimed at HIV-1 eradication. View Publication

HIV persists in latently infected CD4+ T cells during antiretroviral therapy (ART). Immune checkpoint molecules,including PD-1,are preferentially expressed at the surface of persistently infected cells. However,whether PD-1 plays a functional role in HIV latency and reservoir persistence remains unknown. Using CD4+ T cells from HIV-infected individuals,we show that the engagement of PD-1 inhibits viral production at the transcriptional level and abrogates T-cell receptor (TCR)-induced HIV reactivation in latently infected cells. Conversely,PD-1 blockade with the monoclonal antibody pembrolizumab enhances HIV production in combination with the latency reversing agent bryostatin without increasing T cell activation. Our results suggest that the administration of immune checkpoint blockers to HIV-infected individuals on ART may facilitate latency disruption. View Publication

The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function,though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mphis) directs HSC platelet-bias. Mphis from the marrow of aged mice and humans exhibited an activated phenotype,with increased expression of inflammatory signals. Aged marrow Mphis also displayed decreased phagocytic function. Senescent neutrophils,typically cleared by marrow Mphis,were markedly increased in aged mice,consistent with functional defects in Mphi phagocytosis and efferocytosis. In aged mice,Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity,which can process pro-IL1B,was increased in marrow Mphis and neutrophils. Mechanistically,IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice,depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mphis induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mphis and IL1B in the age-associated lineage-skewing of HSCs,and reveals the therapeutic potential of their manipulation as antigeronic targets. View Publication

In life sciences,the material properties of suspended cells have attained significance close to that of fluorescent markers but with the advantage of label-free and unbiased sample characterization. Until recently,cell rheological measurements were either limited by acquisition throughput,excessive post processing,or low-throughput real-time analysis. Real-time deformability cytometry expanded the application of mechanical cell assays to fast on-the-fly phenotyping of large sample sizes,but has been restricted to single material parameters as the Young's modulus. Here,we introduce dynamic real-time deformability cytometry for comprehensive cell rheological measurements at up to 100 cells per second. Utilizing Fourier decomposition,our microfluidic method is able to disentangle cell response to complex hydrodynamic stress distributions and to determine viscoelastic parameters independent of cell shape. We demonstrate the application of our technology for peripheral blood cells in whole blood samples including the discrimination of B- and CD4+ T-lymphocytes by cell rheological properties. View Publication

Due to their complex chemical and physical properties,the effects and mechanisms of action of natural sources of dietary fiber on the intestine are unclear. Pigs are commonly fed high-fiber diets to reduce production costs and non-starch polysaccharide (NSP)-degrading enzymes have been used to increase fiber digestibility. We evaluated the expression of mucin 2 (MUC2),presence of goblet cells,and ileal immune profile of pigs housed individually for 28 days and fed either a low fiber diet based on corn-soybean meal (CSB,n = 9),or two high fiber diets formulated adding 40{\%} corn distillers' dried grains with solubles (DDGS,n = 9) or 30{\%} wheat middlings (WM,n = 9) to CSB-based diet. Pigs were also fed those diets supplemented with a NSP enzymes mix (E) of xylanase,beta-glucanase,mannanase,and galactosidase (n = 8,10,and 9 for CSB+E,DDGS+E and WM+E,respectively). Feeding DDGS and WM diets increased ileal MUC2 expression compared with CSB diet,and this effect was reversed by the addition of enzymes. There were no differences in abundance of goblet cells among treatments. In general,enzyme supplementation increased gene expression and concentrations of IL-1beta,and reduced the concentrations of IL-4,IL-17A and IL-11. The effects of diet-induced cytokines on modulating intestinal MUC2 were assessed in vitro by treating mouse and swine enteroids with 1 ng/ml of IL-4 and IL-1beta. In accordance with previous studies,treatment with Il-4 induced Muc2 and expansion of goblet cells in mouse enteroids. However,swine enteroids did not change MUC2 expression or number of goblet cells when treated with IL-4 or IL-1beta. Our results suggest that mucin and immune profile are regulated by diet in the swine intestine,but by mechanisms different to mouse,emphasizing the need for using appropriate models to study responses to dietary fiber in swine. View Publication

Leukocyte adhesion requires beta2-integrin activation. Resting integrins exist in a bent-closed conformation-i.e.,not extended (E-) and not high affinity (H-)-unable to bind ligand. Fully activated E+H+ integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans. E-H- transitions to E+H+ through E+H- or through E-H+,which binds to ICAMs on the same cell in cis. Spatial patterning of activated integrins is thought to be required for effective arrest,but no high-resolution cell surface localization maps of activated integrins exist. Here,we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest,E-H+ integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern,we blocked integrin binding to ICAMs in cis,which significantly relieved the face-to-face orientation. View Publication

Enterovirus type 71 (EV71) and coxsackievirus A 16 (CA16) are the major pathogens of human hand,foot,and mouth disease (HFMD). In our previous study,intramuscular immunization with the inactivated EV71 vaccine elicited effective immunity,while immunization with the inactivated CA16 vaccine did not. In this report,we focused on innate immune responses elicited by inactivated EV71 and CA16 antigens administered intradermally or intramuscularly. The distributions of the EV71 and CA16 antigens administered intradermally or intramuscularly were not obviously different,but the antigens were detected for a shorter period of time when administered intradermally. The expression levels of NF-kappaB pathway signaling molecules,which were identified as being capable of activating DCs,ILCs,and T cells,were higher in the intradermal group than in the intramuscular group. Antibodies for the EV71 and CA16 antigens colocalized with ILCs and DCs in skin and muscle tissues under fluorescence microscopy. Interestingly,ILC colocalization decreased over time,while DC colocalization increased over time. ELISpot analysis showed that coordination between DCs and ILCs contributed to successful adaptive immunity against vaccine antigens in the skin. EV71 and/or CA16 antigen immunization via the intradermal route was more capable of significantly increasing neutralizing antibody titers and activating specific T cell responses than immunization via the intramuscular route. Furthermore,neonatal mice born to mothers immunized with the EV71 and CA16 antigens were 100{\%} protected against wild-type EV71 or CA16 viral challenge. Together,our results provide new insights into the development of vaccines for HFMD. View Publication

The risk for non-Hodgkin lymphoma (NHL) is markedly increased in persons living with human immunodeficiency virus (HIV) infection,and remains elevated in those on anti-retroviral therapy (cART). Both the loss of immunoregulation of Epstein-Barr virus (EBV) infected cells,as well as chronic B-cell activation,are believed to contribute to the genesis of AIDS-related NHL (AIDS-NHL). However,the mechanisms that lead to AIDS-NHL have not been completely defined. A subset of B cells that is characterized by the secretion of IL10,as well as the expression of the programmed cell death ligand-1 (PD-L1/CD274),was recently described. These PD-L1+ B cells can exert regulatory function,including the dampening of T-cell activation,by interacting with the program cell death protein (PD1) on target cells. The role of PD-L1+ B cells in the development of AIDS-NHL has not been explored. We assessed B cell PD-L1 expression on B cells preceding AIDS-NHL diagnosis in a nested case-control study of HIV+ subjects who went on to develop AIDS-NHL,as well as HIV+ subjects who did not,using multi-color flow cytometry. Archival frozen viable PBMC were obtained from the UCLA Multicenter AIDS Cohort Study (MACS). It was seen that the number of CD19+CD24++CD38++and CD19+PD-L1+cells was significantly elevated in cases 1-4 years prior to AIDS-NHL diagnosis,compared to controls,raising the possibility that these cells may play a role in the etiology of AIDS-NHL. Interestingly,most PD-L1+ expression on CD19+ cells was seen on CD19+CD24++CD38++ cells. In addition,we showed that HIV can directly induce PD-L1 expression on B cells through interaction of virion-associated CD40L with CD40 on B cells. View Publication

BACKGROUND AIMS Inactivating mutations in MYO5B cause microvillus inclusion disease (MVID),but the physiological cause of the diarrhea associated with this disease is unclear. We investigated whether loss of MYO5B results in aberrant expression of apical enterocyte transporters. METHODS We studied alterations in apical membrane transporters in MYO5B-knockout mice,as well as mice with tamoxifen-inducible,intestine-specific disruption of Myo5b (VilCreERT2;Myo5bflox/flox mice) or those not given tamoxifen (controls). Intestinal tissues were collected from mice and analyzed by immunostaining,immunoelectron microscopy,or cultured enteroids were derived. Functions of brush border transporters in intestinal mucosa were measured in Ussing chambers. We obtained duodenal biopsy specimens from individuals with MVID and individuals without MVID (controls) and compared transporter distribution by immunocytochemistry. RESULTS Compared to intestinal tissues from littermate controls,intestinal tissues from MYO5B-knockout mice had decreased apical localization of SLC9A3 (also called NHE3),SLC5A1 (also called SGLT1),aquaporin (AQP) 7,and sucrase isomaltase,and subapical localization of intestinal alkaline phosphatase and CDC42. However,CFTR was present on apical membranes of enterocytes from MYO5B knockout and control mice. Intestinal biopsies from patients with MVID had subapical localization of NHE3,SGLT1,and AQP7,but maintained apical CFTR. After tamoxifen administration,VilCreERT2;Myo5bflox/flox mice lost apical NHE3,SGLT1,DRA,and AQP7,similar to germline MYO5B knockout mice. Intestinal tissues from VilCreERT2;Myo5bflox/flox mice had increased CFTR in crypts and CFTR localized to the apical membranes of enterocytes. Intestinal mucosa from VilCreERT2;Myo5bflox/flox mice given tamoxifen did not have an intestinal barrier defect,based on Ussing chamber analysis,but did have decreased SGLT1 activity and increased CFTR activity. CONCLUSIONS Although trafficking of many apical transporters is regulated by MYO5B,trafficking of CFTR is largely independent of MYO5B. Decreased apical localization of NHE3,SGLT1,DRA,and AQP7 might be responsible for dysfunctional water absorption in enterocytes of patients with MVID. Maintenance of apical CFTR might exacerbate water loss by active secretion of chloride into the intestinal lumen. View Publication

Gastrointestinal (GI) parasites,hookworms in particular,have evolved to cause minimal harm to their hosts,allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed,hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs),exosome-like EVs from different helminths have been characterized and their salient roles in parasite-host interactions have been highlighted. Here,we analyze EVs from the rodent parasite Nippostrongylus brasiliensis,which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids,indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins,including proteins frequently present in exosomes (like tetraspanin,enolase,14-3-3 protein,and heat shock proteins),and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species,many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties,we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6,IL-1$\beta$,IFN$\gamma$,and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast,high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system,such as IBD. View Publication