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Background: Abnormal branched-chained amino acids (BCAA) accumulation in cardiomyocytes is associated with cardiac remodeling in heart failure. Administration of branched-chain α-keto acid dehydrogenase (BCKD) kinase inhibitor BT2 has been shown to reduce cardiac BCAA levels and demonstrated positive effects on cardiac function in a preclinical setting. The current study is focused on evaluating the impact of BT2 on the systemic and cardiac levels of BCAA and their metabolites as well as activities of BCAA catabolic enzymes using a quantitative systems pharmacology model. Methods: The model is composed of an ordinary differential equation system characterizing BCAA consumption with food, disposal in the proteins, reversible branched-chain-amino-acid aminotransferase (BCAT)-mediated transamination to branched-chain keto-acids (BCKA), followed by BCKD-mediated oxidation. Activity of BCKD is regulated by the balance of BCKDK and protein phosphatase 2Cm (PP2Cm) activities, affected by BT2 treatment. Cardiac BCAA levels are assumed to directly affect left ventricular ejection fraction (LVEF). Biochemical characteristics of the enzymes are taken from the public domains, while plasma and cardiac BCAA and BCKA levels in BT2 treated mice are used to inform the model parameters. Results: The model provides adequate reproduction of the experimental data and predicts synchronous BCAA responses in the systemic and cardiac space, dictated by rapid BCAA equilibration between the tissues. The model-based simulations indicate maximum possible effect of BT2 treatment on BCAA reduction to be 40% corresponding to 12% increase in LVEF. Model sensitivity analysis demonstrates strong impact of BCKDK and PP2Cm activities as well as total BCKD and co-substrate levels (glutamate, ketoglutarate and ATP) on BCAA and BCKA levels. Conclusion: Model based simulations confirms using of plasma measurements as a marker of cardiac BCAA changes under BCKDK inhibition. The proposed model can be used for optimization of preclinical study design for novel compounds targeting BCAA catabolism.
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Efficient priming of anti-tumor T cells requires the uptake and presentation of tumor antigens by immunogenic dendritic cells (DCs) and occurs mainly in lymph nodes draining the tumor (tdLNs). However, tumors expand and activate myeloid-derived suppressor cells (MDSCs) that inhibit CTL functions by several mechanisms. While the immune-suppressive nature of the tumor microenvironment is largely documented, it is not known whether similar immune-suppressive mechanisms operate in the tdLNs. In this study, we analyzed MDSC characteristics within tdLNs. We show that, in a metastasis-free context, MO-MDSCs are the dominant MDSC population within tdLNs, that they are highly suppressive and that tumor proximity enhances their recruitment to tdLN via a CCR2/CCL2-dependent pathway. Altogether our results uncover a mechanism by which tumors evade the immune system that involves MDSC-mediated recruitment to the tdLN and the inhibition of T-cell activation even before reaching the highly immunosuppressive tumor microenvironment.
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Células Supresoras de Origen Mieloide/metabolismo , Receptores CCR2/metabolismo , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Femenino , Humanos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/fisiología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Monocitos/metabolismo , Células Mieloides/inmunología , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/fisiología , Neoplasias/inmunología , Receptores CCR2/inmunologíaRESUMEN
PGC1α-Related Coactivator (PRC) is a transcriptional coactivator promoting cytokine expression in vitro in response to mitochondrial injury and oxidative stress, however, its physiological role has remained elusive. Herein we investigate aspects of the immune response function of PRC, first in an in vivo thioacetamide (TAA)-induced mouse model of drug-induced liver injury (DILI), and subsequently in vitro in human monocytes, HepG2, and dendritic (DC) cells. TAA treatment resulted in the dose-dependent induction of PRC mRNA and protein, both of which were shown to correlate with liver injury markers. Conversely, an adenovirus-mediated knockdown of PRC attenuated this response, thereby reducing hepatic cytokine mRNA expression and monocyte infiltration. Subsequent in vitro studies with conditioned media from HepG2 cells overexpressing PRC, activated human monocytes and monocyte-derived DC, demonstrated up to 20% elevated expression of CD86, CD40, and HLA-DR. Similarly, siRNA-mediated knockdown of PRC abolished this response in oligomycin stressed HepG2 cells. A putative mechanism was suggested by the co-immunoprecipitation of Signal Transducer and Activator of Transcription 1 (STAT1) with PRC, and induction of a STAT-dependent reporter. Furthermore, PRC co-activated an NF-κB-dependent reporter, indicating interaction with known major inflammatory factors. In summary, our study indicates PRC as a novel factor modulating inflammation in DILI.
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Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined.Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD.Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21+CXCL13+ (IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs.Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21+CXCL13+ Tfh-like cells. Importantly, the capacity to induce IL-21+ Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX40L (OX40 ligand)-OX40 axis reduced Tfh-cell induction by control lung cDC2s.Conclusions: In COPD lungs, we found lung EBI2+ (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21+ Tfh-like cells, suggesting an involvement of these cells in TLO formation.
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Células Dendríticas/inmunología , Pulmón/citología , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Estructuras Linfoides Terciarias/etiología , Anciano , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Infection of C57Bl/6 mice by pleomorphic African trypanosomes Trypanosoma brucei and T. congolense is characterized by parasitemia waves coupled with the production of systemic levels of TNF. This cytokine is known to control T. brucei growth, but also to contribute to tissue damage, shortening the survival time of infected mice. Using a dominant-negative version of TNF to discriminate between the effects of the membrane-form versus the soluble form of TNF, we show that the second form is involved in neither parasite control nor induction of liver injury. Therefore, soluble TNF is likely not a major contributor to disease outcome. We propose that membrane-bound TNF is responsible for both T. brucei control and host pathology.
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Parasitemia/veterinaria , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/parasitología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Membrana Celular/metabolismo , Ratones , Ratones Endogámicos C57BL , Parasitemia/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/veterinaria , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The bromodomain and plant homeodomain finger-containing (BRPF) family are scaffolding proteins important for the recruitment of histone acetyltransferases of the MYST family to chromatin. Here, we describe NI-57 (16) as new pan-BRPF chemical probe of the bromodomain (BRD) of the BRPFs. Inhibitor 16 preferentially bound the BRD of BRPF1 and BRPF2 over BRPF3, whereas binding to BRD9 was weaker. Compound 16 has excellent selectivity over nonclass IV BRD proteins. Target engagement of BRPF1B and BRPF2 with 16 was demonstrated in nanoBRET and FRAP assays. The binding of 16 to BRPF1B was rationalized through an X-ray cocrystal structure determination, which showed a flipped binding orientation when compared to previous structures. We report studies that show 16 has functional activity in cellular assays by modulation of the phenotype at low micromolar concentrations in both cancer and inflammatory models. Pharmacokinetic data for 16 was generated in mouse with single dose administration showing favorable oral bioavailability.
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Quinolonas/farmacología , Sulfonamidas/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN , Diseño de Fármacos , Estabilidad de Medicamentos , Semivida , Humanos , Ratones , Microsomas Hepáticos/metabolismo , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Quinolonas/administración & dosificación , Quinolonas/síntesis química , Quinolonas/farmacocinética , Relación Estructura-Actividad , Sulfonamidas/administración & dosificación , Sulfonamidas/síntesis química , Sulfonamidas/farmacocinéticaRESUMEN
Various steady state and inflamed tissues have been shown to contain a heterogeneous DC population consisting of developmentally distinct subsets, including cDC1s, cDC2s and monocyte-derived DCs, displaying differential functional specializations. The identification of functionally distinct tumour-associated DC (TADC) subpopulations could prove essential for the understanding of basic TADC biology and for envisaging targeted immunotherapies. We demonstrate that multiple mouse tumours as well as human tumours harbour ontogenically discrete TADC subsets. Monocyte-derived TADCs are prominent in tumour antigen uptake, but lack strong T-cell stimulatory capacity due to NO-mediated immunosuppression. Pre-cDC-derived TADCs have lymph node migratory potential, whereby cDC1s efficiently activate CD8+ T cells and cDC2s induce Th17 cells. Mice vaccinated with cDC2s displayed a reduced tumour growth accompanied by a reprogramming of pro-tumoural TAMs and a reduction of MDSCs, while cDC1 vaccination strongly induces anti-tumour CTLs. Our data might prove important for therapeutic interventions targeted at specific TADC subsets or their precursors.
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Células Dendríticas/inmunología , Neoplasias/inmunología , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Células Cultivadas , Humanos , Inmunoterapia/métodos , Macrófagos/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Monocitos/inmunología , Neoplasias/patología , Neoplasias/terapia , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.
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Macrófagos/metabolismo , Monocitos/metabolismo , Receptores del Factor Estimulante de Colonias/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Microambiente Tumoral/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Diferenciación Celular/fisiología , Polaridad Celular/fisiología , Femenino , Factor Estimulante de Colonias de Macrófagos/inmunología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/patología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Monocitos/patología , Receptores del Factor Estimulante de Colonias/antagonistas & inhibidores , Receptores del Factor Estimulante de Colonias/inmunología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Transducción de SeñalRESUMEN
Monocytes consist of two well-defined subsets, the Ly6C+ and Ly6C- monocytes. Both CD11b+ myeloid cells populations have been proposed to infiltrate tissues during inflammation. While infiltration of Ly6C+ monocytes is an established pathogenic factor during hepatic inflammation, the role of Ly6C- monocytes remains elusive. Mice suffering experimental African trypanosome infection die from systemic inflammatory response syndrome (SIRS) that is initiated by phagocytosis of parasites by liver myeloid cells and culminates in apoptosis/necrosis of liver myeloid and parenchymal cells that reduces host survival. C57BL/6 mice are considered as trypanotolerant to Trypanosoma congolense infection. We have reported that in these animals, IL-10, produced among others by myeloid cells, limits the liver damage caused by pathogenic TNF-producing Ly6C+ monocytes, ensuring prolonged survival. Here, the heterogeneity and dynamics of liver myeloid cells in T. congolense-infected C57/BL6 mice was further dissected. Moreover, the contribution of Ly6C- monocytes to trypanotolerance was investigated. By using FACS analysis and adoptive transfer experiments, we found that the accumulation of Ly6C- monocytes and macrophages in the liver of infected mice coincided with a drop in the pool of Ly6C+ monocytes. Pathogenic TNF mainly originated from Ly6C+ monocytes while Ly6C- monocytes and macrophages were major and equipotent sources of IL-10 within myeloid cells. Moreover, Nr4a1 (Nur77) transcription factor-dependent Ly6C- monocytes exhibited IL-10-dependent and cell contact-dependent regulatory properties contributing to trypanotolerance by suppressing the production of TNF by Ly6C+ monocytes and by promoting the differentiation of the latter cells into macrophages. Thus, Ly6C- monocytes can dampen liver damage caused by an extensive Ly6C+ monocyte-associated inflammatory immune response in T. congolense trypanotolerant animals. In a more general context, Ly6C- or Ly6C+ monocyte targeting may represent a therapeutic approach in liver pathogenicity induced by chronic infection.
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Antígenos Ly/inmunología , Diferenciación Celular , Inflamación/etiología , Hepatopatías/etiología , Macrófagos/inmunología , Monocitos/inmunología , Monocitos/patología , Tripanosomiasis Africana/inmunología , Animales , Apoptosis , Western Blotting , Proliferación Celular , Femenino , Citometría de Flujo , Técnicas para Inmunoenzimas , Inflamación/patología , Interleucina-10/genética , Interleucina-10/metabolismo , Hepatopatías/patología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/inmunología , Células Mieloides/patología , Fagocitosis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Trypanosoma congolense/inmunología , Tripanosomiasis Africana/complicaciones , Tripanosomiasis Africana/parasitología , Células Tumorales CultivadasRESUMEN
Kupffer cells (KCs) are liver resident macrophages which are important for tissue homeostasis and have been implicated in immunogenic, tolerogenic and pathogenic immune reactions depending on the insult. These cells and the biomarkers they express thus represent interesting in vivo sensors for monitoring liver inflammation. In the current study, we explored whether KCs can be monitored non-invasively using single-photon-emission computed tomography (SPECT) with (99m)Tc labeled nanobodies (Nbs) targeting selected biomarkers. Nbs targeting V-set and immunoglobulin domain-containing 4 (Vsig4) or macrophage mannose receptor (MMR) accumulated in the liver of untreated mice. The liver targeting of anti-Vsig4 Nbs, but not anti-MMR Nbs, was blunted upon depletion of macrophages, highlighting specificity of anti-Vsig4 Nbs for liver macrophage imaging. Ex vivo flow cytometry and immunohistochemistry analysis confirmed that anti-Vsig4 Nbs specifically targeted KCs but no other cell types in the liver. Upon induction of acute hepatitis using concanavalin A (ConA), down-regulation of the in vivo imaging signal obtained using anti-Vsig4 Nbs reflected reduction in KC numbers and transient modulation of Vsig4 expression on KCs. Overall, these results indicate that Nbs targeting Vsig4 as molecular imaging biomarker enable non-invasive monitoring of KCs during hepatic inflammation.
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Macrófagos del Hígado/inmunología , Macrófagos del Hígado/metabolismo , Receptores de Complemento/inmunología , Receptores de Complemento/metabolismo , Anticuerpos de Dominio Único/inmunología , Enfermedad Aguda , Animales , Antígenos/inmunología , Antígenos de Superficie/metabolismo , Antígeno CD11b/metabolismo , Recuento de Células , Concanavalina A/efectos adversos , Concanavalina A/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Hepatitis A/inducido químicamente , Hepatitis A/inmunología , Hepatitis A/metabolismo , Inmunofenotipificación , Masculino , Ratones , Imagen Molecular , Fenotipo , Receptores de Complemento/genéticaRESUMEN
Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11b(hi)F4/80(hi)Ly6C(lo) TAM subsets, designated as MHC-II(lo) and MHC-II(hi) TAM, both of which were derived from tumor-infiltrating Ly6C(hi) monocytes. MHC-II(lo) TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II(lo) TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II(hi) counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (PHD2)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-II(lo) TAM subset. The same observation in PHD2(+/+) â PHD2(+/-) bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II(lo) TAM.
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Hipoxia de la Célula/fisiología , Macrófagos/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Animales , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/genética , TranscriptomaRESUMEN
BACKGROUND: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. METHODOLOGY/PRINCIPAL FINDINGS: By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. CONCLUSION: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.
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Arginasa/inmunología , Cinesinas/inmunología , Proteínas Protozoarias/inmunología , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Africana/inmunología , Animales , Arginasa/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Activación Enzimática/genética , Activación Enzimática/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Cinesinas/genética , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Ratones , Ratones Noqueados , Óxido Nítrico/genética , Óxido Nítrico/inmunología , Proteínas Protozoarias/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/patologíaRESUMEN
Tumor growth coincides with an accumulation of myeloid-derived suppressor cells (MDSCs), which exert immune suppression and which consist of two main subpopulations, known as monocytic (MO) CD11b(+) CD115(+) Ly6G(-) Ly6C(high) MDSCs and granulocytic CD11b(+) CD115(-) Ly6G(+) Ly6C(int) polymorphonuclear (PMN)-MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8(+) T-cell activation--including cytokine production, surface marker expression, survival, and cytotoxicity--is currently unclear. Here, employing an in vitro coculture system, we demonstrate that splenic MDSC subsets suppress antigen-driven CD8(+) T-cell proliferation, but differ in their dependency on IFN-γ, STAT-1, IRF-1, and NO to do so. Moreover, MO-MDSC and PMN-MDSCs diminish IL-2 levels, but only MO-MDSCs affect IL-2Rα (CD25) expression and STAT-5 signaling. Unexpectedly, however, both MDSC populations stimulate IFN-γ production by CD8(+) T cells on a per cell basis, illustrating that some T-cell activation characteristics are actually stimulated by MDSCs. Conversely, MO-MDSCs counteract the activation-induced change in CD44, CD62L, CD162, and granzyme B expression, while promoting CD69 and Fas upregulation. Together, these effects result in an altered CD8(+) T-cell adhesiveness to the extracellular matrix and selectins, sensitivity to FasL-mediated apoptosis, and cytotoxicity. Hence, MDSCs intricately influence different CD8(+) T-cell activation events in vitro, whereby some parameters are suppressed while others are stimulated.
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Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Células Mieloides/inmunología , Neoplasias/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos Ly/metabolismo , Apoptosis/inmunología , Antígeno CD11b/metabolismo , Adhesión Celular/inmunología , Línea Celular , Proliferación Celular , Femenino , Granzimas/biosíntesis , Receptores de Hialuranos/biosíntesis , Factor 1 Regulador del Interferón , Interferón gamma/biosíntesis , Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Selectina L/biosíntesis , Lectinas Tipo C/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Óxido Nítrico/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/inmunología , Regulación hacia Arriba , Receptor fas/biosíntesisRESUMEN
Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor-associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a(+) macrophages in M2-associated infectious pathologies. In late-stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll-like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL-4). LPS/dexamethasone/IL-4-induced Ms4a8a(+) BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF-κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL-4-stimulated Ms4a8a(+) BMDMs and in peritoneal macrophages from late-stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2-like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.
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Antígenos CD20/inmunología , Macrófagos/inmunología , Taenia/inmunología , Teniasis/inmunología , Trypanosoma congolense/inmunología , Tripanosomiasis Africana/inmunología , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/inmunología , Arginasa/genética , Arginasa/inmunología , Línea Celular , Inmunidad Innata/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Activación de Macrófagos/inmunología , Macrófagos/parasitología , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN/química , ARN/genética , ARN Mensajero/química , ARN Mensajero/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Organismos Libres de Patógenos Específicos , Teniasis/parasitología , Receptores Toll-Like/agonistas , Tripanosomiasis Africana/parasitologíaRESUMEN
The parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases. Activation of these enzymes requires the dimerization of the catalytic domain and typically occurs under stress. Using a dominant-negative strategy, we found that reducing adenylate cyclase activity by about 50% allowed trypanosome growth but reduced the parasite's ability to control the early innate immune defense of the host. Specifically, activation of trypanosome adenylate cyclase resulting from parasite phagocytosis by liver myeloid cells inhibited the synthesis of the trypanosome-controlling cytokine tumor necrosis factor-α through activation of protein kinase A in these cells. Thus, adenylate cyclase activity of lyzed trypanosomes favors early host colonization by live parasites. The role of adenylate cyclases at the host-parasite interface could explain the expansion and polymorphism of this gene family.
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Adenilil Ciclasas/metabolismo , Inmunidad Innata , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Africana/inmunología , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Animales , Dominio Catalítico , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Interacciones Huésped-Parásitos , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Células Mieloides/inmunología , Parasitemia , Fagocitosis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/metabolismo , Tripanosomiasis Africana/parasitología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/sangreRESUMEN
In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL-4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL-4-dependent E-cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL-4-induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1-deficient macrophages display rather enhanced IL-4-induced polyamine production, suggesting that an Arg1-independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS-induced expression of several proinflammatory genes was increased significantly in polyamine-depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL-4-induced expression of several AAM mediators, they inhibit the LPS-mediated expression of proinflammatory genes in CAMs.
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Arginasa/fisiología , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Interleucina-4/farmacología , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Poliaminas/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Citometría de Flujo , Perfilación de la Expresión Génica , Activación de Macrófagos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor TIE-2RESUMEN
Macrophages display remarkable plasticity, allowing these cells to adapt to changing microenvironments and perform functions as diverse as tissue development and homeostasis, inflammation, pathogen clearance and wound healing. Macrophage activation can be triggered by Th1 cytokines and pathogen-associated or endogenous danger signals, leading to the formation of classically activated or M1 macrophages. On the other hand, anti-inflammatory mediators, including IL-4, IL-10, TGF-ß and M-CSF, induce diverse anti-inflammatory types of macrophages, known under the generic term M2. In human breast carcinomas, tumor-associated macrophage (TAM) density correlates with poor prognosis. In mouse models of breast cancer, eliminating macrophages from the tumor site, either via genetic or therapeutic means, results in retarded tumor progression. Over the years, multiple signals from the mammary tumor microenvironment have been reported to influence the TAM phenotype and TAM have been propagated as anti-inflammatory M2-like cells. Recent developments point to the existence of at least two distinct TAM subpopulations in mammary tumors, based on a differential expression of markers such as CD206 or MHC II and different in vivo behaviour: perivascular, migratory TAM which are less M2-like, and sessile TAM found at tumor-stroma borders and/or hypoxic regions that resemble more M2-like or "trophic" macrophages. Hence, a further refinement of the molecular and functional heterogeneity of TAM is an avenue for further research, with a potential impact on the usefulness of these cells as therapeutic targets.
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Neoplasias de la Mama/patología , Neoplasias de la Mama/fisiopatología , Macrófagos/clasificación , Macrófagos/fisiología , Animales , Neoplasias de la Mama/inmunología , Comunicación Celular , Femenino , Humanos , Mediadores de Inflamación/fisiología , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/patología , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/fisiopatología , Ratones , Modelos Biológicos , PronósticoRESUMEN
A balance between parasite elimination and control of infection-associated pathogenicity is crucial for resistance to African trypanosomiasis. By producing TNF and NO, CD11b(+) myeloid cells with a classical activation status (M1) contribute to parasitemia control in experimental Trypanosoma congolense infection in resistant C57BL/6 mice. However, in these mice, IL-10 is required to regulate M1-associated inflammation, avoiding tissue/liver damage and ensuring prolonged survival. In an effort to dissect the mechanisms behind the anti-inflammatory activity of IL-10 in T. congolense-infected C57BL/6 mice, we show, using an antibody blocking the IL-10 receptor, that IL-10 impairs the accumulation and M1 activation of TNF/iNOS-producing CD11b(+) Ly6C(+) cells in the liver. Using infected IL-10(flox/flox) LysM-Cre(+/+) mice, we show that myeloid cell-derived IL-10 limits M1 activation of CD11b(+) Ly6C(+) cells specifically at the level of TNF production. Moreover, higher production of TNF in infected IL-10(flox/flox) LysM-Cre(+/+) mice is associated with reduced nuclear accumulation of the NF-κB p50 subunit in CD11b(+) M1 cells. Furthermore, in infected p50(-/-) mice, TNF production by CD11b(+) Ly6C(+) cells and liver injury increases. These data suggest that preferential nuclear accumulation of p50 represents an IL-10-dependent anti-inflammatory mechanism in M1-type CD11b(+) myeloid cells that regulates the production of pathogenic TNF during T. congolense infection in resistant C57BL/6 mice.
Asunto(s)
Interleucina-10/inmunología , Células Mieloides/inmunología , Subunidad p50 de NF-kappa B/inmunología , Trypanosoma congolense/inmunología , Tripanosomiasis Africana/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Animales , Western Blotting , Separación Celular , Citometría de Flujo , Interleucina-10/metabolismo , Hígado/citología , Hígado/inmunología , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Subunidad p50 de NF-kappa B/metabolismo , Transducción de Señal/inmunología , Tripanosomiasis Africana/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Mononuclear phagocytes are amongst the most versatile cells of the body, contributing to tissue genesis and homeostasis and safeguarding the balance between pro- and anti-inflammatory reactions. Accordingly, these cells are notoriously heterogeneous, functioning in distinct differentiation forms (monocytes, MDSC, macrophages, DC) and adopting different activation states in response to a changing microenvironment. Accumulating evidence exists that mononuclear phagocytes contribute to all phases of the cancer process. These cells orchestrate the inflammatory events during de novo carcinogenesis, participate in tumor immunosurveillance, and contribute to the progression of established tumors. At the tumor site, cells such as tumor-associated macrophages (TAM) are confronted with different tumor microenvironments, leading to TAM subsets with specialized functions. A better refinement of the molecular and functional heterogeneity of tumor-associated mononuclear phagocytes might pave the way for novel cancer therapies that directly target these tumor-supporting cells.
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Transformación Celular Neoplásica/inmunología , Vigilancia Inmunológica , Neoplasias/inmunología , Fagocitos/inmunología , Transducción de Señal/inmunología , Microambiente Tumoral/inmunología , Animales , Antígenos CD/inmunología , Antígenos CD/metabolismo , Movimiento Celular/inmunología , Transformación Celular Neoplásica/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Progresión de la Enfermedad , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Humanos , Inflamación/inmunología , Macrófagos/citología , Macrófagos/inmunología , Ratones , Monocitos/citología , Monocitos/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Especificidad de Órganos , Fagocitos/clasificación , Fagocitos/citologíaRESUMEN
The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent activation of M1 that produce pathogenic molecules such as TNF and NO contributes to the development of trypanosome infection-associated tissue injury including liver cell necrosis in experimental mouse models. Aiming to identify mechanisms involved in regulation of M1 activity, we have recently documented that during Trypanosoma brucei infection, CD11b(+)Ly6C(+)CD11c(+) TNF and iNOS producing DCs (Tip-DCs) represent the major pathogenic M1 liver subpopulation. By using gene expression analyses, KO mice and cytokine neutralizing antibodies, we show here that the conversion of CD11b(+)Ly6C(+) monocytic cells to pathogenic Tip-DCs in the liver of T. brucei infected mice consists of a three-step process including (i) a CCR2-dependent but CCR5- and Mif-independent step crucial for emigration of CD11b(+)Ly6C(+) monocytic cells from the bone marrow but dispensable for their blood to liver migration; (ii) a differentiation step of liver CD11b(+)Ly6C(+) monocytic cells to immature inflammatory DCs (CD11c(+) but CD80/CD86/MHC-II(low)) which is IFN-gamma and MyD88 signaling independent; and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-II(high)) TNF and NO producing Tip-DCs which is IFN-gamma and MyD88 signaling dependent. Moreover, IL-10 could limit CCR2-mediated egression of CD11b(+)Ly6C(+) monocytic cells from the bone marrow by limiting Ccl2 expression by liver monocytic cells, as well as their differentiation and maturation to Tip-DCs in the liver, showing that IL-10 works at multiple levels to dampen Tip-DC mediated pathogenicity during T. brucei infection. A wide spectrum of liver diseases associates with alteration of monocyte recruitment, phenotype or function, which could be modulated by IL-10. Therefore, investigating the contribution of recruited monocytes to African trypanosome induced liver injury could potentially identify new targets to treat hepatic inflammation in general, and during parasite infection in particular.