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1.
Immunology ; 159(1): 63-74, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573680

RESUMO

Monocyte-derived macrophages (MDMs) generated from peripheral blood monocytes are widely used to model human macrophages for in vitro studies. However, the possible impact of different isolation methods on the resulting MDM phenotype is poorly described. We aimed to investigate the effects of three commonly used monocyte isolation techniques on the resulting MDM phenotype. Plastic adhesion, negative selection, and CD14pos selection were compared. Monocyte-derived macrophages were generated by 5-day culture with macrophage and granulocyte-macrophage colony-stimulating factors. We investigated monocyte and MDM yields, purity, viability, and cell phenotype. CD14pos selection resulted in highest monocyte yield (19·8 × 106 cells, equivalent to 70% of total) and purity (98·7%), compared with negative selection (17·7 × 106 cells, 61% of total, 85·0% purity), and plastic adhesion (6·1 × 106 cells, 12·9% of total, 44·2% purity). Negatively selected monocytes were highly contaminated with platelets. Expression of CD163 and CD14 were significantly lower on CD14pos selection and plastic adhesion monocytes, compared with untouched peripheral blood mononuclear cells. After maturation, CD14pos selection also resulted in the highest MDM purity (98·2%) compared with negative selection (94·5%) and plastic adhesion (66·1%). Furthermore, MDMs from plastic adhesion were M1-skewed (CD80high  HLA-DRhigh  CD163low ), whereas negative selection MDMs were M2-skewed (CD80low  HLA-DRlow  CD163high ). Choice of monocyte isolation method not only significantly affects yield and purity, but also impacts resulting phenotype of cultured MDMs. These differences may partly be explained by the presence of contaminating cells when using plastic adherence or negative selection. Careful considerations of monocyte isolation methods are important for designing in vitro assays on MDMs.


Assuntos
Diferenciação Celular , Separação Celular/métodos , Citometria de Fluxo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/fisiologia , Monócitos/fisiologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores , Adesão Celular , Células Cultivadas , Humanos , Interleucina-6/metabolismo , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Fenótipo , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
2.
Carbohydr Polym ; 229: 115404, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826490

RESUMO

Biomaterial-host interactions significantly affect tissue repair, which is modulated by macrophages. In this study, a polysaccharide, konjac glucomannan (KGM), was acetylated with different degrees of substitution (DS), and the acetylated KGM (AceKGM)-based fibrous membrane was designed to modulate the activity of macrophages for accelerating wound healing. AceKGM was biocompatible and easily dissolved in organic solvents. The adhesion force between Raw264.7 cells and the AceKGM substrate was quantitatively detected by atomic force microscopy (AFM). The enzyme-linked immunosorbent assay (ELISA) results showed that the AceKGM fibrous membrane enhanced macrophage expression of anti-inflammatory and pro-regenerative cytokines, and the DS of AceKGM significantly affected membrane bioactivity. The full-thickness mouse skin wound repair experiments indicated that the AceKGM-containing fibrous membranes significantly accelerated wound healing by promoting re-epithelialization, tissue remodeling, and collagen deposition. In summary, AceKGM-based fibrous membranes have potential as bioactive scaffolds for wound regeneration.


Assuntos
Bandagens , Mananas , Membranas Artificiais , Cicatrização , Acetilação , Animais , Adesão Celular , Lectinas Tipo C/metabolismo , Mananas/química , Lectinas de Ligação a Manose/metabolismo , Camundongos , Células NIH 3T3 , Células RAW 264.7 , Receptores de Superfície Celular/metabolismo
3.
Protein Pept Lett ; 27(1): 60-66, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31362652

RESUMO

BACKGROUND: Cathelicidins are a family of Host Defense Peptides (HDPs), that play an important role in the innate immune response. They exert both broad-spectrum antimicrobial activity against pathogens, and strong immunomodulatory functions that affect the response of innate and adaptive immune cells. OBJECTIVE: The aim of this study was to investigate immunomodulation by the chicken cathelicidin CATH-2 and compare its activities to those of the human cathelicidin LL-37. METHODS: Chicken macrophages and chicken monocytes were incubated with cathelicidins. Activation of immune cells was determined by measuring surface markers Mannose Receptor Ctype 1 (MRC1) and MHC-II. Cytokine production was measured by qPCR and nitric oxide production was determined using the Griess assay. Finally, the effect of cathelicidins on phagocytosis was measured using carboxylate-modified polystyrene latex beads. RESULTS: CATH-2 and its all-D enantiomer D-CATH-2 increased MRC1 and MHC-II expression, markers for antigen presentation, on primary chicken monocytes, whereas LL-37 did not. D-CATH- 2 also increased the MRC1 and MHC-II expression if a chicken macrophage cell line (HD11 cells) was used. In addition, LPS-induced NO production by HD11 cells was inhibited by CATH-2 and D-CATH-2. CONCLUSION: These results are a clear indication that CATH-2 (and D-CATH-2) affect the activation state of monocytes and macrophages, which leads to optimization of the innate immune response and enhancement of the adaptive immune response.


Assuntos
Biomarcadores/metabolismo , Catelicidinas/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Sequência de Aminoácidos , Animais , Apresentação do Antígeno/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Linhagem Celular , Galinhas , Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Imunomodulação/efeitos dos fármacos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
4.
PLoS One ; 14(12): e0226651, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31856198

RESUMO

A single HIV-1 variant establishes infection of the host after sexual contact. Identifying the phenotypic characteristics of these Transmitted Founder (T/F) viruses is important to understand the restriction mechanisms during transmission. Langerhans cells (LCs) are the mucosal dendritic cell subset that has been shown to have a protective role in HIV-1 transmission. Immature LCs efficiently capture and degrade HIV-1 via langerin-mediated restriction. Here we have investigated the capacity of T/F HIV-1 strains to infect mucosal Langerhans cells (LCs). Notably, most T/F variants efficiently infected immature LCs derived from skin and vaginal tissue in contrast to chronic HIV-1 laboratory strains. Next we screened a panel of T/F viruses and their matched 6-month consensus sequence viruses. Interestingly most T/F variants infected immature LCs whereas donor-matched 6-month consensus sequence viruses had lost the ability to infect LCs. However, we also identified 6-month consensus sequence viruses that had retained an ability to infect LCs similar to that of the donor-matched T/F virus. Moreover, some T/F viruses and 6-month consensus sequence viruses were unable to infect immature LCs. Further analyses indicated that T/F viruses are less sensitive to langerin-mediated restriction. These data suggest that T/F HIV-1 variants have the ability to infect immature LCs, which will facilitate transmission.


Assuntos
HIV-1/patogenicidade , Interações Hospedeiro-Patógeno , Células de Langerhans/virologia , Antígenos CD/metabolismo , Células Cultivadas , Células HEK293 , Humanos , Células de Langerhans/imunologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo
5.
J Immunol Res ; 2019: 3161750, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31485459

RESUMO

Rheumatoid arthritis (RA) is a multifactorial autoimmune disease whose main hallmark is inflammation and destruction of the joints. Two cell types within the synovium that play an important role in RA are fibroblast-like synoviocytes (FLS) and macrophages. The latter innate immune cells show a high plasticity in their phenotype and are central in inflammatory processes. Low-dose radiotherapy (LD-RT) with particularly a single dose of 0.5 Gy has been demonstrated to have a positive impact on pain, inflammation, and bone in inflamed joints. We now examined for the first time how LD-RT influences FLS and bone marrow-derived macrophages in co-culture systems of an experimental model of RA to reveal further mechanisms of immune modulatory effects of low and intermediate dose of ionizing radiation. For this, the bone marrow of hTNF-α tg mice was differentiated either with cytokines to obtain key macrophage phenotypes (M0, M1, and M2) or with supernatants (SN) of untreated or irradiated FLS. Flow cytometry analyses were used to analyse the impact of radiation (0.1, 0.5, 1.0, and 2.0 Gy) on the phenotype of macrophages in the presence or absence of SN of FLS. LD-RT had no impact on cytokine-mediated macrophage polarization in M0, M1, or M2 macrophages. However, SN of irradiated FLS particularly reduced CD206 expression on macrophages. Macrophage phenotype was stable when being in contact with SN of nonirradiated FLS, but significantly increased surface expression of CD206 and slightly decreased CD80 and CD86 expression were observed when macrophage themselves were irradiated with 0.5 Gy under these microenvironmental conditions, again highlighting discontinuous dose dependencies in the low and intermediate dose range. One can conclude that FLS-dependent microenvironmental conditions have a slight influence on the modulation of macrophage phenotype under radiation exposure conditions. Future studies are needed to reveal the impact of radiation exposure on the functions of treated macrophages under such microenvironmental conditions.


Assuntos
Artrite Reumatoide/radioterapia , Macrófagos/efeitos da radiação , Sinoviócitos/efeitos da radiação , Animais , Artrite Reumatoide/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Inflamação/imunologia , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/efeitos da radiação , Macrófagos/imunologia , Macrófagos/metabolismo , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Transgênicos , Fenótipo , Doses de Radiação , Receptores de Superfície Celular/metabolismo , Sinoviócitos/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
6.
Am J Chin Med ; 47(6): 1289-1305, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31488032

RESUMO

The roots of Aucklandia lappa have been used in traditional medicine in Asia to treat inflammation and diseases associated with pain, including endometriosis. The aim of this study was to investigate the anti-endometriotic effect of dehydrocostus lactone, an active compound in A. lappa roots, using human endometriotic cells and macrophages stimulated by these cells. Dehydrocostus lactone induced apoptotic cell death in 12Z human endometriotic cells. Dehydrocostus lactone stimulated the activation of caspase-3, -8, and -9, while caspase inhibitors significantly reversed the dehydrocostus lactone-induced cell death in 12Z cells. In addition, dehydrocostus lactone decreased the production of PGE2 and neurotrophins (BDNF, NGF, NT3, and NT4/5), which are regarded as endometriosis-associated pain factors in human endometriotic cells. Moreover, dehydrocostus lactone inhibited the expression of M2 markers (CD206, and Trem-2), IL-10, VEGF, and MMP-2/-9 in endometriosis-associated macrophages (EAMs). Furthermore, dehydrocostus lactone inhibited the Akt and NFκB pathways in both endometriotic cells and EAMs. Taken together, our findings suggest that dehydrocostus lactone, an active compound of A, lappa, has anti-endometriotic activities via induction of apoptosis and downregulation of pain factors in endometriotic cells and inhibition of the alternative activation of EAMs.


Assuntos
Apoptose/efeitos dos fármacos , Endometriose/tratamento farmacológico , Endometriose/imunologia , Endométrio/citologia , Lactonas/farmacologia , Lactonas/uso terapêutico , Ativação de Macrófagos/efeitos dos fármacos , Fitoterapia , Raízes de Plantas/química , Saussurea/química , Sesquiterpenos/farmacologia , Sesquiterpenos/uso terapêutico , Caspases/metabolismo , Linhagem Celular , Dinoprostona/metabolismo , Feminino , Humanos , Interleucina-10/metabolismo , Lactonas/isolamento & purificação , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Medicina Tradicional , Fatores de Crescimento Neural/metabolismo , Receptores de Superfície Celular/metabolismo , Sesquiterpenos/isolamento & purificação , Estimulação Química
7.
Cancer Immunol Immunother ; 68(9): 1455-1465, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31444606

RESUMO

Macrophages have been shown to infiltrate a wide range of malignancies and are often considered to promote tumour survival, growth and spread. However, the source and behaviour of discrete tumour-associated macrophage populations are still poorly understood. Here we show a novel method for the rational development of bone marrow-derived monocytes appropriate for the study of processes which involve the contribution of circulating inflammatory monocytes. We have shown that in response to tumour-conditioned medium, these cells upregulate CD206 and CD115, markers traditionally associated with M2-type macrophages. Treated cells show reduced capacity for cytokine secretion but significantly impact CD4+ and CD8+ T-cell proliferation and polarization. Coculture with conditioned bone marrow-derived monocytes significantly reduced CD4+ T-cell proliferation but increased CD8+ T-cell proliferation and granzyme B expression with significant induction of IFNγ secretion by both CD4+ and CD8+ T cells, indicating that these cells may have a role in promoting anti-cancer immunity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Macrófagos/imunologia , Melanoma/imunologia , Monócitos/imunologia , Neoplasias Cutâneas/imunologia , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Técnicas de Cocultura , Meios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Citotoxicidade Imunológica , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Lectinas de Ligação a Manose/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Células Th2/imunologia
8.
Acta Vet Hung ; 67(2): 183-196, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31238731

RESUMO

The aim of this immunocytochemical study was to compare mannose-binding lectin (MBL) production induced by avian coronavirus in the spleen and caecal tonsil (CT). One-day-old specific-pathogen-free (SPF) chickens were experimentally infected with six QX field isolates and the H120 vaccine strain. In the negative control birds, the spleen was MBL negative, while the CT showed scattered MBL-positive cells in close proximity and within the surface epithelium and germinal centre (GC)-like cell clusters. MBL was detectable in the ellipsoid-associated cells (EACs) and cell clusters in the periarterial lymphoid sheath (PALS) by 7 days post infection (dpi). In both organs, the MBL-positive cells occupy antigen-exposed areas, indicating that GC formation depends on resident precursors of dendritic cells. The majority of MBL-positive EACs express the CD83 antigen, providing evidence that coronavirus infection facilitated the maturation of dendritic cell precursors. Surprisingly, co-localisation of MBL and CD83 was not detectable in the CT. In the spleen (associated with circulation), the EACs producing MBL and expressing CD83 are a common precursor of both follicular (FDC) and interdigitating dendritic cells (IDC). In the CT (gut-associated lymphoid tissue, GALT) the precursors of FDC and IDC are MBL-producing cells and CD83-positive cells, respectively. In the CT the two separate precursors of lymphoid dendritic cells provide some 'autonomy' for the GALT.


Assuntos
Ceco/imunologia , Galinhas , Infecções por Coronavirus/veterinária , Células Dendríticas/metabolismo , Lectinas de Ligação a Manose/metabolismo , Doenças das Aves Domésticas/metabolismo , Baço/imunologia , Animais , Proteínas Aviárias/metabolismo , Infecções por Coronavirus/metabolismo , Gammacoronavirus/fisiologia , Organismos Livres de Patógenos Específicos
9.
Microb Pathog ; 134: 103594, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31199985

RESUMO

Talaromyces marneffei is an increasingly destructive dimorphic fungal pathogen in clinical settings that can cause lethal Talaromycosis. The activation of macrophages is known to be important for host defenses against T. marneffei, and these macrophages are known to be activated in two ways (polarization), known as M1 and M2. We investigated the plasticity of these polarizations, in order to understand if cross-conversion of macrophages may be possible even after they have been programmed. We conducted in vitro experiments using a murine macrophage cell line to investigate the ability of T. marneffei to activate these polarizations. The pre-polarized (M0) macrophage subsets were challenged with LPS as a control, and the sets of M1 markers (iNOS and CD86) and M2 markers (Arg-1 and CD206) were assessed for a possible cross-conversion among M1, M2 and M0 (unstimulated) populations. We found that either conidia or yeast forms of T. marneffei initiate the repression of Arg-1 in M2 cells with no change in the M1 subtype marker molecule iNOS. However, an additional IFN-γ stimulus caused the three macrophage groups to fully exhibit an LPS-induced M2 suppression and a shift to M1 from M0 and M2. We conclude that the conversion of macrophages is required for maintenance of sufficient iNOS production against this organism in the host. The cytokine environment is the key factor that manipulates the plasticity changes among macrophage subtypes. Furthermore, IFN-γ is a crucial host defense factor against pathogenic T. marneffei that has significant therapeutic potential to promote an M1 polarization phenotype.


Assuntos
Interferon gama/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Micoses/imunologia , Talaromyces/imunologia , Animais , Antígeno B7-2/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Citocinas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Camundongos , Micoses/microbiologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Receptores de Superfície Celular/metabolismo
10.
J Immunol Res ; 2019: 6105059, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183389

RESUMO

Sucralose is a noncaloric artificial sweetener that is widely consumed worldwide and has been associated with alteration in glucose and insulin homeostasis. Unbalance in monocyte subpopulations expressing CD11c and CD206 hallmarks metabolic dysfunction but has not yet been studied in response to sucralose. Our goal was to examine the effect of a single sucralose sip on serum insulin and blood glucose and the percentages of classical, intermediate, and nonclassical monocytes in healthy young adults subjected to an oral glucose tolerance test (OGTT). This study was a randomized, placebo-controlled clinical trial. Volunteers randomly received 60 mL water as placebo (n = 20) or 48 mg sucralose dissolved in 60 mL water (n = 25), fifteen minutes prior to an OGTT. Blood samples were individually drawn every 15 minutes for 180 minutes for quantifying glucose and insulin concentrations. Monocyte subsets expressing CD11c and CD206 were measured at -15 and 180 minutes by flow cytometry. As compared to controls, volunteers receiving sucralose exhibited significant increases in serum insulin at 30, 45, and 180 minutes, whereas blood glucose values showed no significant differences. Sucralose consumption caused a significant 7% increase in classical monocytes and 63% decrease in nonclassical monocytes with respect to placebo controls. Pearson's correlation models revealed a strong association of insulin with sucralose-induced monocyte subpopulation unbalance whereas glucose values did not show significant correlations. Sucralose ingestion decreased CD11c expression in all monocyte subsets and reduced CD206 expression in nonclassical monocytes suggesting that sucralose does not only unbalance monocyte subpopulations but also alter their expression pattern of cell surface molecules. This work demonstrates for the first time that a 48 mg sucralose sip increases serum insulin and unbalances monocyte subpopulations expressing CD11c and CD206 in noninsulin-resistant healthy young adults subjected to an OGTT. The apparently innocuous consumption of sucralose should be reexamined in light of these results.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Monócitos/fisiologia , Sacarose/análogos & derivados , Adulto , Glicemia , Antígeno CD11c/metabolismo , Ingestão de Alimentos , Feminino , Teste de Tolerância a Glucose , Voluntários Saudáveis , Humanos , Insulina/metabolismo , Lectinas Tipo C/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Sacarose/administração & dosagem , Adulto Jovem
11.
ACS Appl Mater Interfaces ; 11(25): 22181-22187, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31150201

RESUMO

Triple-negative breast cancer (TNBC) is a devastating disease worldwide, for which targeted imaging and therapeutic agents remain elusive. There has been growing awareness that carbohydrates are valuable as drug candidates and targeting agents for a variety of human diseases, including cancers that overexpress carbohydrate receptors on the cell surface. Here, we develop a two-dimensional (2D) glycocluster by means of simple, stepwise self-assembly for the targeted delivery of theranostic agents to TNBC cells that express mannose receptors (MRs) on the cell surface. Human serum albumin, which contains a variety of hydrophobic pockets capable of accommodating small molecules, was used to simultaneously encapsulate a mannose-based glycoprobe and a commercial photosensitizer (i.e., Ce6). The multicomponent "neoglycoprotein" formed was used to self-assemble with 2D MnO2, producing 2D glycoclusters, which could be selectively internalized by a TNBC cell line (MDA-MB-231) as facilitated by binding to the transmembrane MR. The intracellular degradation of the 2D MnO2 backbone by biothiols then released Ce6 for cell imaging and, subsequently, photodynamic therapy. This study provides insights into the development of carbohydrate-based materials for targeted, stimuli-responsive theranostics of TNBC.


Assuntos
Nanomedicina Teranóstica/métodos , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos/métodos , Feminino , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Fotoquimioterapia/métodos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Espectrometria de Fluorescência/métodos
12.
Int J Nanomedicine ; 14: 3203-3220, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118632

RESUMO

Background: Tumor-associated macrophages (TAMs) are critical in tumor progression and metastasis. Selective targeting of TAMs holds great potential to ameliorate the immunosuppressive tumor microenvironment and enhance the efficacy of antitumor therapy. Various liposomes have been developed to target TAMs via cell-specific surface receptors either to deplete or re-educate TAMs. Since immuno-stimulation often initiates with the interaction of nanocarriers with the innate immunity cells such as macrophages, the intrinsic impact of drug-free liposomes on macrophage activation and polarization via cell interaction is one of the most critical issues in nanomedicine for promoting effective immunotherapy. Methods: In this study, conventional bare liposomes, PEGylated liposomes, and mannosylated liposomes were developed and the cytotoxicity, cellular internalization, immunostimulatory activity, targeting efficiency, antitumor efficacy, and mechanism were evaluated in vitro and in vivo. Results: All liposomes displayed an ideal particle size, good biocompatibility, and controlled release behavior. Mannosylated liposomes exhibited superior in vitro cellular internalization and tumor spheroid penetration with the aid of the mannose receptor-mediated TAMs-targeting effects. In particular, mannosylated liposomes promoted the polarization of both M0 and M2 to the M1 phenotype by enhancing the expression ratio of CD86/CD206 in vitro. Of note, mannosylated liposomes could inhibit G422 glioma tumor growth, which may be attributed to the polarization of TAMs, as evidenced by the reduction in expression level of the TAMs surface marker. Conclusion: These results indicate the potential value of mannosylated liposomes in the design of a rational delivery system to enhance the antitumor immune efficacy of immunomodulators by inducing a shift from the M2 to the M1 phenotype.


Assuntos
Polaridade Celular , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Lectinas de Ligação a Manose/metabolismo , Neoplasias/patologia , Receptores de Superfície Celular/metabolismo , Adjuvantes Imunológicos/farmacologia , Animais , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Cumarínicos/química , Liberação Controlada de Fármacos , Endocitose , Feminino , Humanos , Lipossomos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Células RAW 264.7 , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Tiazóis/química , Distribuição Tecidual , Microambiente Tumoral
14.
Mediators Inflamm ; 2019: 1919538, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31007601

RESUMO

Macrophages are key immune system cells involved in inflammatory processes. Classically activated (M1) macrophages are characterized by strong antimicrobicidal properties, whereas alternatively activated (M2) macrophages are involved in wound healing. Severe inflammation can induce postoperative complications during the perioperative period. Invasive surgical procedures induce polarization to M1 macrophages and associated complications. As perioperative management, it is an important strategy to regulate polarization and functions of macrophages during inflammatory processes. Although propofol has been found to exhibit anti-inflammatory activities in monocytes and macrophages, it is unclear whether propofol regulates the functions of M1 and M2 macrophages during inflammatory processes. This study therefore investigated the effects of propofol on human macrophage polarization. During M1 polarization, propofol suppressed the production of IL-6 and IL-1ß but did not affect TNF-α production. In contrast, propofol did not affect the gene expression of M2 markers, such as IL-10, TGF-ß, and CD206, during M2 polarization. Propofol was similar to the GABAA agonist muscimol in inducing nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) and inhibiting IL-6 and IL-1ß, but not TNF-α, production. Knockdown of Nrf2 using siRNA significantly reduced the effect of propofol on IL-6 and IL-1ß production. These results suggest that propofol prevents inflammatory responses during polarization of human M1 macrophages by suppressing the expression of IL-6 and IL-1ß through the GABAA receptor and the Nrf2-mediated signal transduction pathway.


Assuntos
Citocinas/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Células Cultivadas , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Muscimol/farmacologia , Fator 2 Relacionado a NF-E2/genética , Propofol/farmacologia , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
15.
Immunol Res ; 67(1): 58-69, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30820875

RESUMO

Alternatively activated macrophages (M2) exert anti-inflammatory effects and are crucial for keeping balance between protective and destructive cell-mediated immunity in healing phase of inflammation. Two members of the interferon regulatory factors family, IRF5 and IRF4, are known to promote M1 or M2 phenotype, respectively. Our study aimed to analyse the effectiveness of the M2 differentiation process in vitro (achieved by IL-4 stimulation) and its relationship to the stage of type 1 diabetes mellitus (DM1) in juvenile patients. To identify the basic changes in M2 phenotype, we examined the expression of the surface CD206, CD14, CD86 molecules, intracellular IRF4 and IRF5 transcription factors as well as IL-10 and TNFα intracellular production. Ten newly diagnosed (ND-DM1) and ten long-standing (LS-DM1) patients were enrolled into the study. The control group consisted of six children. We observed a significantly higher number of unpolarised CD206+CD14+ cells in the M2 cultures of DM1 subjects when compared to healthy ones. Examined cells presented common features with M1 macrophages (high levels of the CD14/CD86/IRF5 markers); however, they were weak TNFα producers in ND-DM1 patients. For the first time, we have revealed dysregulated IRF4/IRF5 axis in the analysed subpopulation derived from diabetic patients. Additionally, monocytes of ND-DM1 children were still able to differentiate into regulatory IL-10+ M2 macrophages, while this process was highly limited in LS-DM1 patients. Summarising, we suggest that the M2 polarisation process is less effective in DM1 patients than in healthy subjects and it may vary depending on the stage of disease. It can be concluded that in vitro differentiated M2 macrophages may be used in the future as inflammatory inhibitors for adoptive therapy experiments in ND-DM1 subjects.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Interleucina-10/metabolismo , Macrófagos/imunologia , Adolescente , Diferenciação Celular , Células Cultivadas , Criança , Feminino , Humanos , Fatores Reguladores de Interferon/metabolismo , Interleucina-4/metabolismo , Lectinas Tipo C/metabolismo , Receptores de Lipopolissacarídeos , Masculino , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Células Th2/imunologia
16.
J Immunol Res ; 2019: 5364632, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729137

RESUMO

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025-33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Melanoma/terapia , Proteínas Virais/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células , Epitopos de Linfócito T/imunologia , Imunoterapia/métodos , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Lectinas de Ligação a Manose/metabolismo , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Virais/administração & dosagem
17.
BMC Microbiol ; 19(1): 33, 2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30736731

RESUMO

BACKGROUND: Lactobacillus mucosae DPC 6426 has previously demonstrated potentially cardio-protective properties, in the form of dyslipidaemia and hypercholesterolemia correction in an apolipoprotein-E deficient mouse model. This study aims to characterise the manner in which this microbe may modulate host bile pool composition and immune response, in the context of cardiovascular disease. Lactobacillus mucosae DPC 6426 was assessed for bile salt hydrolase activity and specificity. The microbe was compared against several other enteric strains of the same species, as well as a confirmed bile salt hydrolase-active strain, Lactobacillus reuteri APC 2587. RESULTS: Quantitative bile salt hydrolase assays revealed that enzymatic extracts from Lactobacillus reuteri APC 2587 and Lactobacillus mucosae DPC 6426 demonstrate the greatest activity in vitro. Bile acid profiling of porcine and murine bile following incubation with Lactobacillus mucosae DPC 6426 confirmed a preference for hydrolysis of glyco-conjugated bile acids. In addition, the purified exopolysaccharide and secretome of Lactobacillus mucosae DPC 6426 were investigated for immunomodulatory capabilities using RAW264.7 macrophages. Gene expression data revealed that both fractions stimulated increases in interleukin-6 and interleukin-10 gene transcription in the murine macrophages, while the entire secretome was necessary to increase CD206 transcription. Moreover, the exopolysaccharide elicited a dose-dependent increase in nitric oxide and interleukin-10 production from RAW264.7 macrophages, concurrent with increased tumour necrosis factor-α secretion at all doses. CONCLUSIONS: This study indicates that Lactobacillus mucosae DPC 6426 modulates both bile pool composition and immune system tone in a manner which may contribute significantly to the previously identified cardio-protective phenotype.


Assuntos
Amidoidrolases/biossíntese , Bile/metabolismo , Imunomodulação , Lactobacillus/enzimologia , Lactobacillus/imunologia , Macrófagos/imunologia , Animais , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/microbiologia , Glicosiltransferases/metabolismo , Hidrólise , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Lactobacillus reuteri/enzimologia , Lectinas Tipo C/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Lectinas de Ligação a Manose/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Polissacarídeos Bacterianos/farmacologia , Células RAW 264.7 , Receptores de Superfície Celular/metabolismo , Suínos , Fator de Necrose Tumoral alfa/metabolismo
18.
Oncogene ; 38(23): 4512-4526, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30742098

RESUMO

Liver fibrosis and fibrosis-associated hepatocarcinogenesis are driven by chronic inflammation and are leading causes of morbidity and death worldwide. SYK signaling regulates critical processes in innate and adaptive immunity, as well as parenchymal cells. We discovered high SYK expression in the parenchymal hepatocyte, hepatic stellate cell (HSC), and the inflammatory compartments in the fibrotic liver. We postulated that targeting SYK would mitigate hepatic fibrosis and oncogenic progression. We found that inhibition of SYK with the selective small molecule inhibitors Piceatannol and PRT062607 markedly protected against toxin-induced hepatic fibrosis, associated hepatocellular injury and intra-hepatic inflammation, and hepatocarcinogenesis. SYK inhibition resulted in increased intra-tumoral expression of the p16 and p53 but decreased expression of Bcl-xL and SMAD4. Further, hepatic expression of genes regulating angiogenesis, apoptosis, cell cycle regulation, and cellular senescence were affected by targeting SYK. We found that SYK inhibition mitigated both HSC trans-differentiation and acquisition of an inflammatory phenotype in T cells, B cells, and myeloid cells. However, in vivo experiments employing selective targeted deletion of SYK indicated that only SYK deletion in the myeloid compartment was sufficient to confer protection against fibrogenic progression. Targeting SYK promoted myeloid cell differentiation into hepato-protective TNFαlow CD206hi phenotype downregulating mTOR, IL-8 signaling and oxidative phosphorylation. Collectively, these data suggest that SYK is an attractive target for experimental therapeutics in treating hepatic fibrosis and oncogenesis.


Assuntos
Cirrose Hepática/patologia , Células Mieloides/metabolismo , Transdução de Sinais , Quinase Syk/metabolismo , Animais , Carcinogênese , Carcinoma Hepatocelular/metabolismo , Transdiferenciação Celular , Cicloexilaminas/farmacologia , Feminino , Fibrose , Células Estreladas do Fígado/citologia , Humanos , Interleucina-8/metabolismo , Lectinas Tipo C/metabolismo , Fígado/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Fosforilação Oxidativa , Fenótipo , Pirimidinas/farmacologia , Receptores de Superfície Celular/metabolismo , Estilbenos/farmacologia , Quinase Syk/antagonistas & inibidores , Transcriptoma
19.
Cell Physiol Biochem ; 52(1): 40-56, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790504

RESUMO

BACKGROUND/AIMS: Therapies using stem/progenitor cells have been experimentally and clinically investigated to regenerate damaged hearts. Substance-P (SP) induces bone marrow (BM) stem cell mobilization and suppresses inflammation in ischemic injuries. This study investigated the role of SP in BM stem cell mobilization and immune responses for tissue repair after ischemic-reperfusion injury (IRI), in comparison with that of granulocyte colony-stimulating factor (GCSF). METHODS: SP was intravenously injected into IRI rats and its affect was evaluated by determining colony forming efficiency, immune cell/ cytokine profiles, histological changes, and heart function through echocardiography. RESULTS: In the rat cardiac IRI model, SP suppressed IRI-mediated tumor necrosis factor-α induction, but increased the levels of interleukin-10, CD206+ monocytes, and regulatory T cells in the blood; reduced myocardial apoptosis at day 1 post-IRI; and markedly stimulated colony forming unit (CFU)-e and (CFU)-f cell mobilization. Efficacy of SP in the recovery of cardiac function after IRI was demonstrated by increased cardiac contractility, accompanied by reduced infarction sizes and fibrosis, and increased revascularization of vessels covered with alpha smooth muscle actin. These effects of SP were confirmed in an acute myocardial infarction (AMI) model. All effects mediated by SP were superior to those mediated by GCSF. CONCLUSION: Systemic injection of SP decreased early inflammatory responses and promoted stem cell mobilization, leading to a compact vasculature and improved cardiac function in cardiac IRI and AMI.


Assuntos
Mobilização de Células-Tronco Hematopoéticas , Infarto do Miocárdio , Traumatismo por Reperfusão Miocárdica , Substância P/farmacocinética , Animais , Fator Estimulador de Colônias de Granulócitos/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Interleucina-10/metabolismo , Lectinas Tipo C/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
Biochem Biophys Res Commun ; 511(2): 356-362, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30791981

RESUMO

The endoplasmic reticulum-Golgi intermediate compartment protein-53 (ERGIC-53, aka LMAN1), which cycles between the endoplasmic reticulum (ER) and Golgi, is a known cargo receptor for a number of soluble proteins. However, whether LMAN1 plays a role as a trafficking factor in the central nervous system is largely unknown. Here, we determined the role of LMAN1 on endogenous protein levels of the Cys-loop superfamily of neuroreceptors, including gamma-aminobutyric acid type A receptors (GABAARs), 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptors, and nicotinic acetylcholine receptors (nAChRs). Knockdown of LMAN1 reduces the surface trafficking of endogenous ß3 subunits of GABAARs in mouse hypothalamic GT1-7 neurons. Furthermore, Western blot analysis of brain homogenates from LMAN1 knockout mice demonstrated that loss of LMAN1 decreases the total protein levels of 5HT3A receptors and γ2 subunits of GABAARs. LMAN1 knockout regulates the ER proteostasis network by upregulating ERP44 without changing calnexin levels. Interestingly, despite the critical role of the glycan-binding function of LMAN1 in its other known cargo clients, LMAN1 interacts with GABAARs in a glycan-independent manner. In summary, LMAN1 is a trafficking factor for certain neuroreceptors in the central nervous system. This is the first report of LMAN1 function in membrane protein trafficking.


Assuntos
Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Receptores de GABA-A/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular , Humanos , Lectinas de Ligação a Manose/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Transporte Proteico
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