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Antibody-based CD47 blockade aims to activate macrophage phagocytosis of tumor cells. However, macrophages possess a high degree of phenotype heterogeneity that likely influences phagocytic capacity. In murine models, proinflammatory (M1) activation increases macrophage phagocytosis of tumor cells, but in human models, results have been conflicting. Here, we investigated the effects of proinflammatory polarization on the phagocytic response of human monocyte-derived macrophages in an in vitro model. Using both flow cytometry-based and fluorescence live-cell imaging-based phagocytosis assays, we observed that mouse monoclonal anti-CD47 antibody (B6H12) induced monocyte-derived macrophage phagocytosis of cancer cells in vitro. Proinflammatory (M1) macrophage polarization with IFN-γ+LPS resulted in a severe reduction in phagocytic response to CD47 blockade. This reduction coincided with increased expression of the antiphagocytic membrane proteins LILRB1 and Siglec-10 but was not rescued by combination blockade of the corresponding ligands. However, matrix metalloproteinase inhibitors (TAPI-0 or GM6001) partly restored response to CD47 blockade in a dose-dependent manner. In summary, these data suggest that proinflammatory (M1) activation reduces phagocytic response to CD47 blockade in human monocyte-derived macrophages.
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Antígeno CD47 , Macrófagos , Fagocitosis , Humanos , Antígeno CD47/inmunología , Antígeno CD47/metabolismo , Antígeno CD47/antagonistas & inhibidores , Macrófagos/inmunología , Macrófagos/metabolismo , Fagocitosis/inmunología , Activación de Macrófagos/inmunología , Activación de Macrófagos/efectos de los fármacos , Inflamación/inmunología , Anticuerpos Monoclonales/farmacología , Ratones , Animales , Línea Celular Tumoral , Neoplasias/inmunología , Interferón gamma/metabolismo , Interferón gamma/inmunologíaRESUMEN
Alpha-synuclein (α-syn) aggregation and immune activation represent hallmark pathological events in Parkinson's disease (PD). The PD-associated immune response encompasses both brain and peripheral immune cells, although little is known about the immune proteins relevant for such a response. We propose that the upregulation of CD163 observed in blood monocytes and in the responsive microglia in PD patients is a protective mechanism in the disease. To investigate this, we used the PD model based on intrastriatal injections of murine α-syn pre-formed fibrils in CD163 knockout (KO) mice and wild-type littermates. CD163KO females revealed an impaired and differential early immune response to α-syn pathology as revealed by immunohistochemical and transcriptomic analysis. After 6 months, CD163KO females showed an exacerbated immune response and α-syn pathology, which ultimately led to dopaminergic neurodegeneration of greater magnitude. These findings support a sex-dimorphic neuroprotective role for CD163 during α-syn-induced neurodegeneration.
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Macrophages are important innate immune cells with the ability to adapt their phenotype to environmental cues. Research on human macrophages often uses monocyte-derived macrophages cultured in vitro, but it is unclear if culture medium affects macrophage phenotype. The objective of this study was to determine the impact of culture medium composition on monocyte-derived macrophage phenotype. Monocyte-derived macrophages were generated in different formulations of culture media (RPMI 1640, DMEM, MEM, McCoy's 5a and IMDM). Viability, yield and cell size were monitored, and RT-qPCR, flow cytometry or ELISA was used to compare levels of phenotype markers (CD163, CD206, CD80, TNFα, IL-10, SIRPα, LILRB1 and Siglec-10). Yield, cell size, gene expression, membrane protein levels and release of soluble proteins were all affected by changes in culture medium composition. The most pronounced effects were observed after culture in DMEM, which lacks the non-essential amino acids asparagine, aspartic acid, glutamic acid and proline. Supplementation of DMEM with non-essential amino acids either fully or partly reversed most effects of DMEM on macrophage phenotype. The results suggest culture medium composition and amino acid availability affect the phenotype of human monocyte-derived macrophages cultured in vitro.
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Aminoácidos , Macrófagos , Humanos , Medios de Cultivo/metabolismo , Fenotipo , Aminoácidos/metabolismo , Citometría de Flujo/métodos , MonocitosRESUMEN
More than 80% of human cancers originate in epithelial tissues. Loss of epithelial cell characteristics are hallmarks of tumor development. Receptor-mediated endocytosis is a key function of absorptive epithelial cells with importance for cellular and organismal homeostasis. LRP2 (megalin) is the largest known endocytic membrane receptor and is essential for endocytosis of various ligands in specialized epithelia, including the proximal tubules of the kidney, the thyroid gland, and breast glandular epithelium. However, the role and regulation of LRP2 in cancers that arise from these tissues has not been delineated. Here, we examined the expression of LRP2 across 33 cancer types in The Cancer Genome Atlas. As expected, the highest levels of LRP2 were found in cancer types that arise from LRP2-expressing absorptive epithelial cells. However, in a subset of tumors from these cancer types, we observed epigenetic silencing of LRP2. LRP2 expression showed a strong inverse correlation to methylation of a specific CpG site (cg02361027) in the first intron of the LRP2 gene. Interestingly, low expression of LRP2 was associated with poor patient outcome in clear cell renal cell carcinoma, papillary renal cell carcinoma, mesothelioma, papillary thyroid carcinoma, and invasive breast carcinoma. Furthermore, loss of LRP2 expression was associated with dedifferentiated histological and molecular subtypes of these cancers. These observations now motivate further studies on the functional role of LRP2 in tumors of epithelial origin and the potential use of LRP2 as a cancer biomarker.
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Objective: The programmed death-1 (PD-1) pathway is essential for maintaining self-tolerance and plays an important role in autoimmunity, including rheumatoid arthritis (RA). Here, we investigated how membrane-bound and soluble (s)PD-1 influence bone homeostasis during chronic inflammation, exemplified in RA. Methods: Bone mineral density and bone microstructure were examined in PD-1 and PD-L1 knockout (KO) mice and compared with wild-type (WT) mice. Receptor activator of nuclear factor kappa-B ligand (RANKL) was measured in serum, and the expression examined on activated bone marrow cells. Osteoclast formation was examined in cells from murine spleen and bone marrow and from human synovial fluid cells. sPD-1 was measured in chronic and early (e)RA patients and correlated to markers of disease activity and radiographic scores. Results: PD-1 and PD-L1 KO mice showed signs of osteoporosis. This was supported by a significantly reduced trabecular bone volume fraction and deteriorated microstructure, as well as increased osteoclast formation and an increased RANKL/OPG ratio. The recombinant form of sPD-1 decreased osteoclast formation in vitro, but was closely associated with disease activity markers in eRA patients. Sustained elevated sPD-1 levels indicated ongoing inflammation and were associated with increased radiographic progression. Conclusion: The PD-1 pathway is closely associated with bone homeostasis, and lacking members of this pathway causes a deteriorated bone structure. The immunological balance in the microenvironment determines how the PD-1 pathway regulates osteoclast formation. In eRA patients, sPD-1 may serve as a biomarker, reflecting residual but clinically silent disease activity and radiographic progression.
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Artritis Reumatoide , Osteoclastos , Animales , Artritis Reumatoide/metabolismo , Antígeno B7-H1 , Biomarcadores , Humanos , Inflamación , Ratones , Osteoclastos/metabolismo , Receptor de Muerte Celular Programada 1/genéticaRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent condition that has been linked to high-fructose corn syrup consumption with induction of hepatic de novo lipogenesis (DNL) as the suggested central mechanism. Feeding diets very high in fructose (> 60%) rapidly induce several features of NAFLD in rodents, but similar diets have not yet been applied in larger animals, such as pigs. With the aim to develop a large animal NAFLD model, we analysed the effects of feeding a high-fructose (HF, 60% w/w) diet for four weeks to castrated male Danish Landrace-York-Duroc pigs. HF feeding upregulated expression of hepatic DNL proteins, but levels were low compared with adipose tissue. No steatosis or hepatocellular ballooning was seen on histopathological examination, and plasma levels of transaminases were similar between groups. Inflammatory infiltrates and the amount of connective tissue was slightly elevated in liver sections from fructose-fed pigs, which was corroborated by up-regulation of macrophage marker expression in liver homogenates. Supported by RNA-profiling, quantitative protein analysis, histopathological examination, and biochemistry, our data suggest that pigs, contrary to rodents and humans, are protected against fructose-induced steatosis by relying on adipose tissue rather than liver for DNL.
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Tejido Adiposo/metabolismo , Carbohidratos de la Dieta/efectos adversos , Fructosa/efectos adversos , Lipogénesis , Enfermedad del Hígado Graso no Alcohólico/etiología , Animales , Carbohidratos de la Dieta/administración & dosificación , Modelos Animales de Enfermedad , Fructosa/administración & dosificación , Humanos , Hígado/metabolismo , Hígado/patología , Masculino , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Enfermedad del Hígado Graso no Alcohólico/patología , Especificidad de la Especie , Sus scrofaRESUMEN
Atherosclerosis is an inflammatory disease characterized by the accumulation of macrophages in the vessel wall. Macrophages depend on their polarization to exert either pro-inflammatory or anti-inflammatory effects. Macrophages of the anti-inflammatory phenotype express high levels of CD163, a scavenger receptor for the hemoglobin-haptoglobin complex. CD163 can also bind to the pro-inflammatory cytokine TWEAK. Using ApoE-deficient or ApoE/CD163 double-deficient mice we aim to investigate the involvement of CD163 in atherosclerosis development and its capacity to neutralize the TWEAK actions. ApoE/CD163 double-deficient mice displayed a more unstable plaque phenotype characterized by an increased lipid and macrophage content, plaque size, and pro-inflammatory cytokine expression. In vitro experiments demonstrated that the absence of CD163 in M2-type macrophages-induced foam cell formation through upregulation of CD36 expression. Moreover, exogenous TWEAK administration increased atherosclerotic lesion size, lipids, and macrophages content in ApoE-/- /CD163-/- compared with ApoE-/- /CD163+/+ mice. Treatment with recombinant CD163 was able to neutralize the proatherogenic effects of TWEAK in ApoE/CD163 double-deficient mice. Recombinant CD163 abolished the pro-inflammatory actions of TWEAK on vascular smooth muscle cells, decreasing NF-kB activation, cytokines and metalloproteinases expression, and macrophages migration. In conclusion, CD163-expressing macrophages serve as a protective mechanism to prevent the deleterious effects of TWEAK on atherosclerotic plaque development and progression.
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Antígenos CD/fisiología , Antígenos de Diferenciación Mielomonocítica/fisiología , Aterosclerosis/patología , Citocina TWEAK/metabolismo , Células Espumosas/patología , Macrófagos/patología , Placa Aterosclerótica/patología , Receptores de Superficie Celular/fisiología , Animales , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Citocinas/metabolismo , Femenino , Células Espumosas/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Noqueados para ApoE , Placa Aterosclerótica/etiología , Placa Aterosclerótica/metabolismoRESUMEN
OBJECTIVE: Active rheumatoid arthritis (RA) is accompanied by increased appendicular and axial bone loss, closely associated to the degree of inflammation. The programmed death-1 (PD-1) pathway is important for maintaining peripheral tolerance, and its ligand PD-L2 has recently been associated with bone morphogenetic protein activity. Here, we report that PD-L2 plays a central role in RA osteoimmunology. METHODS: Femoral bone mineral density (BMD) and trabecular bone microstructure were evaluated by micro-CT in wild type (WT) and PD-L2-/- mice. Osteoclasts were generated from RA synovial fluid mononuclear cells and peripheral blood monocytes. The effects of recombinant PD-L2, was evaluated by tartrate-resistant acid phosphatase (TRAP) activity and the development of bone erosions in the presence of anti-citrullinated protein antibodies (ACPA). Plasma soluble (s)PD-L2 levels were measured in patients with early (e)RA (n â= â103) treated with methotrexate alone or in combination with the TNF inhibitor Adalimumab. RESULTS: PD-L2-/- mice had a decreased BMD and deteriorated trabecular bone microstructure that was not related to the RANKL/OPG pathway. PD-L2 decreased TRAP activity in osteoclasts and decreased ACPA-induced erosions. In the RA synovial membrane PD-L2 was highly expressed especially in the lining layer and plasma sPD-L2 levels were increased in eRA patients and decreased with treatment. One-year sPD-L2 correlated inversely with erosive progression two years after treatment initiation with methotrexate and placebo. CONCLUSION: PD-L2 regulates bone homeostasis in RA. Our findings provide new insight into the relationship between the immune system and bone homeostasis, and suggest a potential therapeutic target for limiting inflammatory bone loss in RA.
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The macrophage is a key cell in the pro- and anti-inflammatory response including that of the inflammatory microenvironment of malignant tumors. Much current drug development in chronic inflammatory diseases and cancer therefore focuses on the macrophage as a target for immunotherapy. However, this strategy is complicated by the pleiotropic phenotype of the macrophage that is highly responsive to its microenvironment. The plasticity leads to numerous types of macrophages with rather different and, to some extent, opposing functionalities, as evident by the existence of macrophages with either stimulating or down-regulating effect on inflammation and tumor growth. The phenotypes are characterized by different surface markers and the present review describes recent progress in drug-targeting of the surface marker CD163 expressed in a subpopulation of macrophages. CD163 is an abundant endocytic receptor for multiple ligands, quantitatively important being the haptoglobin-hemoglobin complex. The microenvironment of inflammation and tumorigenesis is particular rich in CD163+ macrophages. The use of antibodies for directing anti-inflammatory (e.g., glucocorticoids) or tumoricidal (e.g., doxorubicin) drugs to CD163+ macrophages in animal models of inflammation and cancer has demonstrated a high efficacy of the conjugate drugs. This macrophage-targeting approach has a low toxicity profile that may highly improve the therapeutic window of many current drugs and drug candidates.
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Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Inflamación/genética , Terapia Molecular Dirigida , Neoplasias/genética , Receptores de Superficie Celular/genética , Humanos , Inflamación/patología , Inflamación/terapia , Macrófagos/metabolismo , Macrófagos/patología , Neoplasias/patología , Neoplasias/terapia , Microambiente Tumoral/genéticaRESUMEN
The scavenger receptor CD163 is highly expressed in macrophages in sites of chronic inflammation where it has a not yet defined role. Here we have investigated development of collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) in CD163-deficient C57BL/6 mice. Compared to wild-type mice, the CIA in CD163-deficient mice had a several-fold higher arthritis score with early onset, prolonged disease and strongly enhanced progression. Further, the serum anti-collagen antibody isotypes as well as the cytokine profiles and T cell markers in the inflamed joints revealed that CD163-deficient mice after 52 days had a predominant Th2 response in opposition to a predominant Th1 response in CD163+/+ mice. Less difference in disease severity between the CD163+/+ and CD163-/- mice was seen in the CAIA model that to a large extent induces arthritis independently of T-cell response and endogenous Th1/Th2 balance. In conclusion, the present set of data points on a novel strong anti-inflammatory role of CD163.
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Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Receptores de Superficie Celular/deficiencia , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/inmunología , Artritis Experimental/sangre , Artritis Experimental/diagnóstico , Artritis Experimental/patología , Artritis Reumatoide/sangre , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/patología , Colágeno Tipo II/inmunología , Citocinas/sangre , Citocinas/inmunología , Citocinas/metabolismo , Progresión de la Enfermedad , Humanos , Articulaciones/inmunología , Articulaciones/patología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Índice de Severidad de la Enfermedad , Líquido Sinovial/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Balance Th1 - Th2 , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
After subarachnoid haemorrhage, prolonged exposure to toxic extracellular haemoglobin occurs in the brain. Here, we investigate the role of haemoglobin neurotoxicity in vivo and its prevention. In humans after subarachnoid haemorrhage, haemoglobin in cerebrospinal fluid was associated with neurofilament light chain, a marker of neuronal damage. Most haemoglobin was not complexed with haptoglobin, an endogenous haemoglobin scavenger present at very low concentration in the brain. Exogenously added haptoglobin bound most uncomplexed haemoglobin, in the first 2 weeks after human subarachnoid haemorrhage, indicating a wide therapeutic window. In mice, the behavioural, vascular, cellular and molecular changes seen after human subarachnoid haemorrhage were recapitulated by modelling a single aspect of subarachnoid haemorrhage: prolonged intrathecal exposure to haemoglobin. Haemoglobin-induced behavioural deficits and astrocytic, microglial and synaptic changes were attenuated by haptoglobin. Haptoglobin treatment did not attenuate large-vessel vasospasm, yet improved clinical outcome by restricting diffusion of haemoglobin into the parenchyma and reducing small-vessel vasospasm. In summary, haemoglobin toxicity is of clinical importance and preventable by haptoglobin, independent of large-vessel vasospasm.
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The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.
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Actomiosina/metabolismo , Apolipoproteína L1/química , Apolipoproteína L1/genética , Apolipoproteínas L/metabolismo , Enfermedades Renales/metabolismo , Mutación/genética , Secuencia de Aminoácidos , Apolipoproteína L1/orina , Calcio/metabolismo , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Enfermedades Renales/orina , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Fenotipo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Podocitos/ultraestructura , Poli I-C/farmacología , Canales de Potasio/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: Vitamin D3 possesses anti-inflammatory and modulatory properties in addition to its role in calcium and phosphate homeostasis. Upon activation, macrophages (M) can initiate and sustain pro-inflammatory cytokine production in inflammatory disorders and play a pathogenic role in certain cancers. PURPOSE: The main purpose of this study was to encapsulate and specifically target calcitriol to macrophages and investigate the anti-inflammatory properties of calcitriol in vitro and in vivo. METHODS: In this study we have designed and developed near-infrared calcitriol PEGylated nanoparticles (PEG-LNP(Cal)) using a microfluidic mixing technique and modified lipid nanoparticles (LNPs) to target the M specific endocytic receptor CD163. We have investigated LNP cellular uptake and anti-inflammatory effect in LPS-induced M in vitro by flow cytometry, confocal microscopy and gene expression analyses. LNP pharmacodynamics, bio-distribution and organ specific LNP accumulation was also investigated in mice in vivo. RESULTS: In vitro, we observed the specific uptake of PEG-LNP(Cal)-hCD163 in human M, which was significantly higher than the non-specific uptake of control PEG-LNP(Cal)-IgG(h) in M. Pretreatment with encapsulated calcitriol was able to attenuate intracellular TNF-expression, and M surface marker HLA-DR expression more efficiently than free calcitriol in LPS-induced M in vitro. Encapsulated calcitriol diminished mRNA gene levels of TNF-, NF-B, MCP-1 and IL-6, while upregulating IL-10. TNF- and IL-6 protein secretion also decreased. In mice, an in vivo pharmacodynamic study of PEG-LNP(Cal) showed a rapid clearance of IgG and CD163 modified LNPs compared to PEG-LNP(Cal). Antibody modified PEG-LNP(Cal) accumulated in the liver, spleen and kidney, whereas unmodified PEG-LNP(Cal) accumulation was only observed in the liver. CONCLUSION: Our results show that calcitriol can be effectively targeted to M. Our data confirms the anti-inflammatory properties of calcitriol and this may be a potential way to deliver high dose bioactive calcitriol to M during inflammation in vivo.
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Antiinflamatorios/farmacología , Calcitriol/administración & dosificación , Calcitriol/farmacología , Lípidos/química , Macrófagos/metabolismo , Nanopartículas/química , Animales , Anticuerpos/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Calcitriol/farmacocinética , Quimiocinas/metabolismo , Composición de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Tamaño de la Partícula , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/metabolismo , Distribución Tisular/efectos de los fármacosRESUMEN
Tumor-associated macrophages (TAMs) are of major importance in cancer-related immune suppression, and tumor infiltration by CD163pos TAMs is associated with poor outcome in most human cancers. Therefore, therapeutic strategies for reprogramming TAMs from a tumor-supporting (M2-like) phenotype towards a tumoricidal (M1-like) phenotype are of great interest. Activation of the transcription factor STAT3 within the tumor microenvironment is associated with worse prognosis, and STAT3 activation promotes the immunosuppressive phenotype of TAMs. Therefore, we aimed to develop a drug for inhibition of STAT3 specifically within human TAMs by targeting the endocytic CD163 scavenger receptor, which is highly expressed on TAMs. Here, we report the first data on a CD163-targeted STAT3-inhibitory drug consisting of corosolic acid (CA) packaged within long-circulating liposomes (LCLs), which are CD163-targeted by modification with monoclonal anti-CD163 antibodies (αCD163)-CA-LCL-αCD163. We show, that activation of STAT3 (by phosphorylation) was inhibited by CA-LCL-αCD163 specifically within CD163pos cells, with minor effect on CD163neg cells. Furthermore, CA-LCL-αCD163 inhibited STAT3-regulated gene expression of IL-10, and increased expression of TNFα, thus indicating a pro-inflammatory effect of the drug on human macrophages. This M1-like reprogramming at the mRNA level was confirmed by significantly elevated levels of pro-inflammatory cytokines (IFNγ, IL-12, TNFα, IL-2) in the culture medium. Since liposomes are attractive vehicles for novel anti-cancer drugs, and since direct TAM-targeting may decrease adverse effects of systemic inhibition of STAT3, the present results encourage future investigation of CA-LCL-αCD163 in the in vivo setting.
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Macrófagos/fisiología , Monocitos/fisiología , Receptores de Superficie Celular/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Triterpenos/administración & dosificación , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Células Cultivadas , Citocinas/biosíntesis , Composición de Medicamentos , Humanos , Interleucina-10/farmacología , Liposomas , Macrófagos/efectos de los fármacos , Monocitos/efectos de los fármacos , Triterpenos/toxicidadRESUMEN
Tenofovir disoproxil fumarate (TDF) is a commonly used antiretroviral drug for HIV, rarely causing Fanconi syndrome and acute kidney injury. We retrospectively analyzed the clinico pathological presentation of 20 cases of tenofovir-induced tubulopathy, and investigated the renal expression of the megalin and cubilin proteins, as well as the mitochondrial respiratory chain activity. Estimated glomerular filtration rate (eGFR) before TDF exposure was 92 ml/min/1.73m2, decreasing to 27.5 ml/min/1.73m2 at the time of biopsy, with 30% of patients requiring renal replacement therapy. Proximal tubular expression of megalin and cubilin was altered in 19 and 18 cases, respectively, whereas it was preserved in patients exposed to TDF without proximal tubular dysfunction and in HIV-negative patients with acute tubular necrosis. Loss of megalin/cubilin was correlated with low eGFR and high urine retinol binding protein at the time of biopsy, low eGFR at last follow-up, and was more severe in patients with multifactorial toxicity. Patients with additional nephrotoxic conditions promoting tenofovir accumulation showed a lower eGFR at presentation and at last follow-up, and more severe lesions of acute tubular necrosis, than those with isolated tenofovir toxicity. Altered mitochondrial COX activity in proximal tubules was observed and may be an early cellular alteration in tenofovir nephrotoxicity. In conclusion, altered megalin/cubilin expression represents a distinctive feature in tenofovir-induced tubulopathy, and its severity is correlated with urine retinol binding protein loss and is associated with a poor renal prognosis. Concomitant exposure to other nephrotoxic conditions severely impacts the renal presentation and outcome.
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Lesión Renal Aguda/inducido químicamente , Adenina/análogos & derivados , Antirretrovirales/efectos adversos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/biosíntesis , Mitocondrias/efectos de los fármacos , Ácidos Fosforosos/efectos adversos , Receptores de Superficie Celular/biosíntesis , Adenina/efectos adversos , Adulto , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Humanos , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/efectos de los fármacos , Masculino , Persona de Mediana Edad , Receptores de Superficie Celular/efectos de los fármacos , Estudios RetrospectivosRESUMEN
Hemoglobin (Hb) toxicity precipitates secondary brain damage following intracerebral hemorrhage (ICH). CD163 is an anti-inflammatory Hb scavenger receptor and CD163-positive macrophages/microglia locally accumulate post-bleed, yet no studies have investigated the role of CD163 after ICH. ICH was induced in wildtype and CD163-/- mice and various anatomical and functional outcomes were assessed. At 3 d, CD163-/- mice have 43.4 ± 5.0% (p = 0.0002) and 34.8 ± 3.4% (p = 0.0003) less hematoma volume and tissue injury, respectively. Whereas, at 10 d, CD163-/- mice have 49.2 ± 15.0% larger lesions (p = 0.0385). An inflection point was identified, where CD163-/- mice perform better on neurobehavioral testing and have less mortality before 4 d, but increased mortality and worse function after 4 d (p = 0.0389). At 3 d, CD163-/- mice have less Hb, iron, and blood-brain barrier dysfunction, increased astrogliosis and neovascularization, and no change in heme oxygenase 1 (HO1) expression. At 10 d, CD163-/- mice have increased iron and VEGF immunoreactivity, but no significant change in HO1 or astrogliosis. These novel findings reveal that CD163 deficiency has distinct temporal influences following ICH, with early beneficial properties but delayed injurious effects. While it is unclear why CD163 deficiency is initially beneficial, the late injurious effects are consistent with the key anti-inflammatory role of CD163 in the recovery phase of tissue damage.
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Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Hemorragia Cerebral/genética , Receptores de Superficie Celular/genética , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Barrera Hematoencefálica , Encéfalo/patología , Hemorragia Cerebral/metabolismo , Hemorragia Cerebral/psicología , Gliosis/etiología , Gliosis/genética , Hematoma/genética , Hematoma/patología , Hemoglobinas/metabolismo , Hierro/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica/genética , Desempeño Psicomotor , Receptores de Superficie Celular/metabolismo , Recuperación de la FunciónRESUMEN
INTRODUCTION: Extracellular vesicles (EVs) have been recognized as route of communication in the microenvironment. They transfer proteins and microRNAs (miRNAs) between cells, and possess immunoregulatory properties. However, their role in immune-mediated diseases remains to be elucidated. We hypothesized a role for EVs in the rheumatoid arthritis (RA) joint, potentially involving the development of T cell exhaustion and transfer of the co-inhibitory receptor programmed death 1 (PD-1). METHODS: Synovial fluid mononuclear cells (SFMCs) and peripheral blood mononuclear cells (PBMCs) from RA patients were investigated for PD-1 and other markers of T cell inhibition. EVs were isolated from RA plasma and synovial fluid. In addition, healthy control (HC) and RA PBMCs and SFMCs were cultured to produce EVs. These were isolated and investigated by immunogold electron microscopy (EM) and also co-cultured with lymphocytes and PD-1 negative cells to investigate their functions. Finally, the miRNA expression profiles were assessed in EVs isolated from RA and HC cell cultures. RESULTS: Cells from the RA joint expressed several T cell co-inhibitory receptors, including PD-1, TIM-3, and Tigit. ELISA demonstrated the presence of PD-1 in EVs from RA plasma and synovial fluid. Immunogold EM visualized PD-1 expression by EVs. Co-culturing lymphocytes and the PD-1 negative cell line, U937 with EVs resulted in an induction of PD-1 on these cells. Moreover, EVs from RA PBMCs increased proliferation in lymphocytes when co-cultured with these. All EVs contained miRNAs associated with PD-1 and other markers of T cell inhibition and the content was significantly lower in EVs from RA PBMCs than HC PBMCs. Stimulation of the cells increased the miRNA expression. However, EVs isolated from stimulated RA SFMCs did not change their miRNA expression profile to the same extend. CONCLUSION: EVs carrying both the PD-1 receptor and miRNAs associated with T cell inhibition were present in RA cell cultures. Upon stimulation, these miRNAs failed to be upregulated in EVs from RA SFMCs. This was in line with increased expression of T cell co-inhibitory markers on SFMCs. In conclusion, we suggest EVs to play a significant role in the RA microenvironment, potentially favoring the progression of T cell exhaustion.
RESUMEN
Matrix metalloproteinases (MMPs) are regulated at multiple transcriptional and post-transcriptional levels, among which receptor-mediated endocytic clearance. We previously showed that low-density lipoprotein receptor-related protein-1 (LRP-1) mediates the clearance of a complex between the zymogen form of MMP-2 (proMMP-2) and tissue inhibitor of metalloproteinases, TIMP-2, in HT1080 human fibrosarcoma cells. Here we show that, in BN16 rat yolk sac cells, proMMP-2:TIMP-2 complex is endocytosed through a distinct LRP member, megalin/LRP-2. Addition of receptor-associated protein (RAP), a natural LRP antagonist, caused accumulation of endogenous proMMP-2 and TIMP-2 in conditioned media. Incubation with RAP also inhibited membrane binding and cellular uptake of exogenous iodinated proMMP-2:TIMP-2. Moreover, antibodies against megalin/LRP-2, but not against LRP-1, inhibited binding of proMMP-2:TIMP-2 to BN16 cell surface. BIAcore analysis confirmed direct interaction between the complex and megalin/LRP-2. Conditional renal invalidation of megalin/LRP-2 in mice resulted in accumulation of proMMP-2 and TIMP-2 in their urine, highlighting the physiological relevance of the binding. We conclude that megalin/LRP-2 can efficiently mediate cell-surface binding and endocytosis of proMMP-2:TIMP-2 complex. Therefore megalin/LRP-2 can be considered as a new actor in regulation of MMP-2 activity, an enzyme crucially involved in many pathological processes.
Asunto(s)
Precursores Enzimáticos/metabolismo , Gelatinasas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Medios de Cultivo Condicionados , Precursores Enzimáticos/orina , Gelatinasas/orina , Riñón/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Metaloproteinasa 2 de la Matriz/orina , Ratones , Ratones Transgénicos , Complejos Multiproteicos/metabolismo , Unión Proteica , Transporte de Proteínas , Ratas , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
Scavenger receptors constitute a large family of proteins that are structurally diverse and participate in a wide range of biological functions. These receptors are expressed predominantly by myeloid cells and recognize a diverse variety of ligands including endogenous and modified host-derived molecules and microbial pathogens. There are currently eight classes of scavenger receptors, many of which have multiple names, leading to inconsistencies and confusion in the literature. To address this problem, a workshop was organized by the United States National Institute of Allergy and Infectious Diseases, National Institutes of Health, to help develop a clear definition of scavenger receptors and a standardized nomenclature based on that definition. Fifteen experts in the scavenger receptor field attended the workshop and, after extensive discussion, reached a consensus regarding the definition of scavenger receptors and a proposed scavenger receptor nomenclature. Scavenger receptors were defined as cell surface receptors that typically bind multiple ligands and promote the removal of nonself or altered-self targets. They often function by mechanisms that include endocytosis, phagocytosis, adhesion, and signaling that ultimately lead to the elimination of degraded or harmful substances. Based on this definition, nomenclature and classification of these receptors into 10 classes were proposed. This classification was discussed at three national meetings and input from participants at these meetings was requested. The following manuscript is a consensus statement that combines the recommendations of the initial workshop and incorporates the input received from the participants at the three national meetings.
Asunto(s)
Receptores Depuradores/clasificación , Receptores Depuradores/fisiología , Animales , Endocitosis , Humanos , Ligandos , Ratones , National Institute of Allergy and Infectious Diseases (U.S.)/normas , Fagocitosis , Receptores Inmunológicos/fisiología , Receptores Depuradores de Clase A/fisiología , Transducción de Señal , Terminología como Asunto , Estados UnidosRESUMEN
Increased consumption of high-caloric carbohydrates contributes substantially to endemic non-alcoholic fatty liver disease in humans, covering a histological spectrum from fatty liver to steatohepatitis. Hypercaloric intake and lipogenetic effects of fructose and endotoxin-driven activation of liver macrophages are suggested to be essential to disease progression. In the present study, we show that a low dose of an anti-CD163-IgG-dexamethasone conjugate targeting the hemoglobin scavenger receptor CD163 in Kupffer cells and other M2-type macrophages has a profound effect on liver inflammatory changes in rats on a high-fructose diet. The diet induced severe non-alcoholic steatohepatitis (NASH)-like changes within a few weeks but the antibody-drug conjugate strongly reduced inflammation, hepatocyte ballooning, fibrosis, and glycogen deposition. Non-conjugated dexamethasone or dexamethasone conjugated to a control IgG did not have this effect but instead exacerbated liver lipid accumulation. The low-dose anti-CD163-IgG-dexamethasone conjugate displayed no apparent systemic side effects. In conclusion, macrophage targeting by antibody-directed anti-inflammatory low-dose glucocorticoid therapy seems to be a promising approach for safe treatment of fructose-induced liver inflammation.