RESUMEN
Neutrophils are the most abundant immune cells found in actively inflamed joints of patients with rheumatoid arthritis (RA), and most animal models for RA depend on neutrophils for the induction of joint inflammation. Exogenous IL-4 and IL-13 protect mice from antibody-mediated joint inflammation, although the mechanism is not understood. Neutrophils display a very strong basal expression of STAT6, which is responsible for signaling following exposure to IL-4 and IL-13. Still, the role of IL-4 and IL-13 in neutrophil biology has not been well studied. This can be explained by the low neutrophil surface expression of the IL-4 receptor α-chain (IL-4Rα), essential for IL-4- and IL-13-induced STAT6 signaling. Here we identify that colony stimulating factor 3 (CSF3), released during acute inflammation, mediates potent STAT3-dependent neutrophil IL-4Rα up-regulation during sterile inflammatory conditions. We further demonstrate that IL-4 limits neutrophil migration to inflamed joints, and that CSF3 combined with IL-4 or IL-13 results in a prominent neutrophil up-regulation of the inhibitory Fcγ receptor (FcγR2b). Taking these data together, we demonstrate that the IL-4 and CSF3 pathways are linked and play important roles in regulating proinflammatory neutrophil behavior.
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Artritis/metabolismo , Interleucina-4 , Infiltración Neutrófila/fisiología , Neutrófilos/metabolismo , Receptores de IgG/metabolismo , Animales , Modelos Animales de Enfermedad , Interleucina-4/genética , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Inhibition of microsomal prostaglandin E synthase-1 (mPGES-1) results in decreased production of proinflammatory PGE2 and can lead to shunting of PGH2 into the prostaglandin D2 (PGD2)/15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) pathway. 15dPGJ2 forms Michael adducts with thiol-containing biomolecules such as GSH or cysteine residues on target proteins and is thought to promote resolution of inflammation. We aimed to elucidate the biosynthesis and metabolism of 15dPGJ2 via conjugation with GSH, to form 15dPGJ2-glutathione (15dPGJ2-GS) and 15dPGJ2-cysteine (15dPGJ2-Cys) conjugates and to characterize the effects of mPGES-1 inhibition on the PGD2/15dPGJ2 pathway in mouse and human immune cells. Our results demonstrate the formation of PGD2, 15dPGJ2, 15dPGJ2-GS, and 15dPGJ2-Cys in RAW264.7 cells after lipopolysaccharide stimulation. Moreover, 15dPGJ2-Cys was found in lipopolysaccharide-activated primary murine macrophages as well as in human mast cells following stimulation of the IgE-receptor. Our results also suggest that the microsomal glutathione S-transferase 3 is essential for the formation of 15dPGJ2 conjugates. In contrast to inhibition of cyclooxygenase, which leads to blockage of the PGD2/15dPGJ2 pathway, we found that inhibition of mPGES-1 preserves PGD2 and its metabolites. Collectively, this study highlights the formation of 15dPGJ2-GS and 15dPGJ2-Cys in mouse and human immune cells, the involvement of microsomal glutathione S-transferase 3 in their biosynthesis, and their unchanged formation following inhibition of mPGES-1. The results encourage further research on their roles as bioactive lipid mediators.
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Cisteína , Prostaglandinas , Ratones , Humanos , Animales , Lipopolisacáridos/metabolismo , Mastocitos , Prostaglandina-E Sintasas/metabolismo , Macrófagos/metabolismo , Ciclooxigenasa 2/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Prostaglandina D2/farmacologíaRESUMEN
Proteins subjected to post-translational modifications, such as citrullination, carbamylation, acetylation or malondialdehyde (MDA)-modification are targeted by autoantibodies in seropositive rheumatoid arthritis (RA). Epidemiological and experimental studies have both suggested the pathogenicity of such humoral autoimmunity, however, molecular mechanisms triggered by anti-modified protein antibodies have remained to be identified. Here we describe in detail the pathways induced by anti-MDA modified protein antibodies that were obtained from synovial B cells of RA patients and that possessed robust osteoclast stimulatory potential and induced bone erosion in vivo. Anti-MDA antibodies boosted glycolysis in developing osteoclasts via an FcγRI, HIF-1α and MYC-dependent mechanism and subsequently increased oxidative phosphorylation. Osteoclast development required robust phosphoglyceride and triacylglyceride biosynthesis, which was also enhanced by anti-MDA by modulating citrate production and expression of the glycerol-3-phosphate dehydrogenase 1 (GPD1) and glycerol-3-phosphate acyltransferase 2 (GPAT2) genes. In summary, we described novel metabolic pathways instrumental for osteoclast differentiation, which were targeted by anti-MDA antibodies, accelerating bone erosion, a central component of RA pathogenesis.
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Artritis Reumatoide , Autoanticuerpos , Humanos , Malondialdehído , LípidosRESUMEN
Dysregulated T-cell activation is a hallmark of several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). The lymphocyte cytosolic protein 2 (LCP2), also known as SLP-76, is essential for the development and activation of T cells. Despite the critical role of LCP2 in T-cell activation and the need for developing drugs that modify T-cell activation, no LCP2 inhibitors have been developed. This can be explained by the "undruggable" nature of LCP2, lacking a structure permissive to standard small molecule inhibitor modalities. Here, we explored an alternative drug modality, developing antisense oligonucleotides (ASOs) targeting LCP2 mRNAs, and evaluated its activity in modulating T-cell activation. We identified a set of 3' UTR targeting LCP2 ASOs, which knocked down LCP2 in a human T-cell line and primary human T cells and found that these suppressed T-cell receptor mediated activation. We also found that the ASOs suppressed FcεR1-mediated mast cell activation, in line with the role of LCP2 in mast cells. Taken together, our data provide examples of how immunomodulatory ASOs that interfere with undruggable targets can be developed and propose that such drug modalities can be used to treat autoimmune diseases.
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Enfermedades Autoinmunes , Oligonucleótidos Antisentido , Línea Celular , Humanos , Activación de Linfocitos , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Linfocitos TRESUMEN
BACKGROUND: Atherosclerosis progression is modulated by interactions with the adaptive immune system. Humoral immunity can help protect against atherosclerosis formation; however, the existence, origin, and function of putative atherogenic antibodies are controversial. How such atherosclerosis-promoting antibodies could affect the specific composition and stability of plaques, as well as the vasculature generally, remains unknown. METHODS: We addressed the overall contribution of antibodies to atherosclerosis plaque formation, composition, and stability in vivo (1) with mice that displayed a general loss of antibodies, (2) with mice that had selectively ablated germinal center-derived IgG production, or (3) through interruption of T-B-cell interactions and further studied the effects of antibody deficiency on the aorta by transcriptomics. RESULTS: Here, we demonstrate that atherosclerosis-prone mice with attenuated plasma cell function manifest reduced plaque burden, indicating that antibodies promote atherosclerotic lesion size. However, the composition of the plaque was altered in antibody-deficient mice, with an increase in lipid content and decreases in smooth muscle cells and macrophages, resulting in an experimentally validated vulnerable plaque phenotype. Furthermore, IgG antibodies enhanced smooth muscle cell proliferation in vitro in an Fc receptor-dependent manner, and antibody-deficient mice had decreased neointimal hyperplasia formation in vivo. These IgG antibodies were shown to be derived from germinal centers, and mice genetically deficient for germinal center formation had strongly reduced atherosclerosis plaque formation. mRNA sequencing of aortas revealed that antibodies are required for the sufficient expression of multiple signal-induced and growth-promoting transcription factors and that aortas undergo large-scale metabolic reprograming in their absence. Using an elastase model, we demonstrated that absence of IgG results in an increased severity of aneurysm formation. CONCLUSIONS: We propose that germinal center-derived IgG antibodies promote the size and stability of atherosclerosis plaques, through promoting arterial smooth muscle cell proliferation and maintaining the molecular identity of the aorta. These results could have implications for therapies that target B cells or B-T-cell interactions because the loss of humoral immunity leads to a smaller but less stable plaque phenotype.
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Aorta/inmunología , Enfermedades de la Aorta/inmunología , Aterosclerosis/inmunología , Centro Germinal/inmunología , Inmunoglobulina G/inmunología , Placa Aterosclerótica , Animales , Antígenos CD19/genética , Antígenos CD19/metabolismo , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Centro Germinal/metabolismo , Inmunoglobulina G/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Factor 1 de Unión al Dominio 1 de Regulación Positiva/deficiencia , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Rotura Espontánea , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.
Asunto(s)
Infecciones por Virus de Epstein-Barr , Enfermedad de Hodgkin , Linfocitos B , Células Cultivadas , Herpesvirus Humano 4 , Enfermedad de Hodgkin/genética , HumanosRESUMEN
Motivation: Genome editing using versions of the bacterial CRISPR/Cas9 system can be used to probe the function of selected genes in any organism. Green Listed is a web-based tool that rapidly designs custom CRISPR screens targeting sets of genes defined by the user. It could thus be used to design screens targeting for example all genes differentially expressed during a specific stimuli or all genes related to a specific pathway or function, as well as to generate targeted secondary screens following a large-scale screen. Availability and Implementation: The software, including a demo function as well as explanatory texts and videos, is available through greenlisted.cmm.ki.se . Contact: fredrik.wermeling@ki.se.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Programas InformáticosRESUMEN
Autoimmune diseases are characterized by pathogenic immune responses to self-antigens. In systemic lupus erythematosus (SLE), many self-antigens are found in apoptotic cells (ACs), and defects in removal of ACs from the body are linked to a risk for developing SLE. This includes pathological memory that gives rise to disease flares. In this study, we investigated how memory to AC-derived self-antigens develops and the contribution of self-memory to the development of lupus-related pathology. Multiple injections of ACs without adjuvant into wild-type mice induce a transient primary autoimmune response without apparent anti-nuclear Ab reactivity or kidney pathology. Interestingly, as the transient Ab response reached baseline, a single boost injection fully recalled the immune response to ACs, and this memory response was furthermore transferable into naive mice. Additionally, the memory response contains elements of pathogenicity, accompanied by selective memory to selective Ags. Thus, we provide evidence for a selective self-memory that underlies progression of the response to self-antigens with implications for SLE development therapy.
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Apoptosis/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Memoria Inmunológica/inmunología , Lupus Eritematoso Sistémico/inmunología , Animales , Lupus Eritematoso Sistémico/terapia , Ratones , Ratones Endogámicos C57BLRESUMEN
Marginal zone macrophages (MZM) are strategically located in the spleen, lining the marginal sinus where they sense inflammation and capture Ag from the circulation. One of the receptors expressed by MZM is scavenger receptor macrophage receptor with collagenous structure (MARCO), which has affinity for modified self-antigens. In this article, we show that engagement of MARCO on murine macrophages induces extracellular ATP and loss of CD21 and CD62L on marginal zone B cells. Engagement of MARCO also leads to reduction of Ag transport by marginal zone B cells and affects the subsequent immune response. This study highlights a novel function for MZM in regulating Ag transport and activation, and we suggest that MARCO-dependent ATP release regulates this through shedding of CD21 and CD62L. Because systemic lupus erythematosus patients were shown to acquire autoantibodies against MARCO, this highlights a mechanism that could affect a patient's ability to combat infections.
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Antígenos/metabolismo , Linfocitos B/inmunología , Macrófagos/fisiología , Receptores de Complemento 3d/fisiología , Bazo/inmunología , Inmunidad Adaptativa , Adenosina Trifosfato/metabolismo , Animales , Selectina L/fisiología , Ratones , Receptores Inmunológicos/fisiologíaRESUMEN
High-dose intravenous immunoglobulin is a widely used therapeutic preparation of highly purified immunoglobulin G (IgG) antibodies. It is administered at high doses (1-2 grams per kilogram) for the suppression of autoantibody-triggered inflammation in a variety of clinical settings. This anti-inflammatory activity of intravenous immunoglobulin is triggered by a minor population of IgG crystallizable fragments (Fcs), with glycans terminating in α2,6 sialic acids (sFc) that target myeloid regulatory cells expressing the lectin dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN; also known as CD209). Here, to characterize this response in detail, we generated humanized DC-SIGN mice (hDC-SIGN), and demonstrate that the anti-inflammatory activity of intravenous immunoglobulin can be recapitulated by the transfer of bone-marrow-derived sFc-treated hDC-SIGN(+) macrophages or dendritic cells into naive recipients. Furthermore, sFc administration results in the production of IL-33, which, in turn, induces expansion of IL-4-producing basophils that promote increased expression of the inhibitory Fc receptor FcγRIIB on effector macrophages. Systemic administration of the T(H)2 cytokines IL-33 or IL-4 upregulates FcγRIIB on macrophages, and suppresses serum-induced arthritis. Consistent with these results, transfer of IL-33-treated basophils suppressed induced arthritic inflammation. This novel DC-SIGN-T(H)2 pathway initiated by an endogenous ligand, sFc, provides an intrinsic mechanism for maintaining immune homeostasis that could be manipulated to provide therapeutic benefit in autoimmune diseases.
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Inmunoglobulinas Intravenosas/inmunología , Inflamación/inmunología , Células Th2/inmunología , Animales , Artritis/tratamiento farmacológico , Artritis/inmunología , Artritis/patología , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Basófilos/efectos de los fármacos , Basófilos/inmunología , Basófilos/metabolismo , Médula Ósea , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Cristalización , Células Dendríticas/citología , Células Dendríticas/inmunología , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Inmunoglobulinas Intravenosas/química , Inmunoglobulinas Intravenosas/metabolismo , Inmunoglobulinas Intravenosas/farmacología , Inflamación/tratamiento farmacológico , Interleucina-33 , Interleucina-4/inmunología , Interleucina-4/metabolismo , Interleucinas/inmunología , Interleucinas/metabolismo , Interleucinas/farmacología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Lectinas Tipo C/metabolismo , Ligandos , Macrófagos/citología , Macrófagos/inmunología , Ratones , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Receptores de Superficie Celular/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Células Th2/efectos de los fármacosRESUMEN
The anti-inflammatory drug high-dose intravenous immunoglobulin, widely used to suppress inflammation, depends on a specific α-2,6-sialylated glycoform of IgG Fc to induce Interleukin 4 (IL-4) and Signal Transducer and Activator of Transcription 6 (STAT6) signaling for its activity. Here we show that anti-inflammatory activities of IL-4 can be attributed to the direct action of this cytokine on myeloid effector cells, depending on their expression of the IL-4 receptor alpha chain (IL-4Rα/CD124). However, in their basal state, these cells express low levels of IL-4Rα and would not be expected to result in significant signaling compared with other cell populations. This apparent paradox can be explained by the observation that during inflammation, triggered by a variety of stimuli (including autoantibodies, adjuvants, and TLR ligands), IL-4Rα is up-regulated specifically on these cells, priming them for STAT6 signaling. The regulation is mediated by a soluble, proteinase K-sensitive factor, released to the circulation by bone marrow-derived, non-B/non-T cells found in several organs, including the lungs, and fat. We propose that this regulation is part of a homeostatic mechanism to limit excessive inflammation and tissue damage. High-dose intravenous immunoglobulin thus exploits an endogenous feedback loop, general to inflammation, that could be further targeted for therapeutic purposes.
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Regulación de la Expresión Génica/inmunología , Homeostasis/inmunología , Inmunoglobulinas Intravenosas/farmacología , Inflamación/inmunología , Receptores de Superficie Celular/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/inmunología , Animales , Western Blotting , Articulaciones del Carpo/inmunología , Citometría de Flujo , Técnicas Histológicas , Inmunoglobulinas Intravenosas/metabolismo , Inflamación/tratamiento farmacológico , Ratones , Ratones Noqueados , Células Mieloides , Reacción en Cadena de la Polimerasa , Receptores de Superficie Celular/genética , Receptores de IgG/metabolismo , Transducción de Señal/genética , Estadísticas no ParamétricasRESUMEN
BACKGROUND/AIMS: Inflammation is a major and critical component of the lung pathology in the hereditary disease cystic fibrosis. The molecular mechanisms of chronic inflammation in cystic fibrosis require definition. METHODS: We used several genetic mouse models to test a role of iNKT cells and ceramide in pulmonary inflammation of cystic fibrosis mice. Inflammation was determined by the pulmonary cytokine profil and the abundance of inflammatory cells in the lung. RESULTS: Here we provide a new concept how inflammation in the lung of individuals with cystic fibrosis is initiated. We show that in cystic fibrosis mice the mutation in the Cftr gene provokes a significant up-regulation of iNKT cells in the lung. Accumulation of iNKT cells serves to control autoimmune disease, which is triggered by a ceramide-mediated induction of cell death in CF organs. Autoimmunity becomes in particular overt in cystic fibrosis mice lacking iNKT cells and although suppression of the autoimmune response by iNKT cells is beneficial, IL-17(+) iNKT cells attract macrophages and neutrophils to CF lungs resulting in chronic inflammation. Genetic deletion of iNKT cells in cystic fibrosis mice prevents inflammation in CF lungs. CONCLUSION: Our data demonstrate an important function of iNKT cells in the chronic inflammation affecting cystic fibrosis lungs. iNKT cells suppress the auto-immune response induced by ceramide-mediated death of epithelial cells in CF lungs, but also induce a chronic pulmonary inflammation.
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Células Asesinas Naturales/inmunología , Animales , Autoanticuerpos/metabolismo , Autoinmunidad , Linfocitos B/inmunología , Linfocitos B/metabolismo , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Modelos Animales de Enfermedad , Interleucina-17/metabolismo , Células Asesinas Naturales/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CFTR , Neumonía/metabolismo , Neumonía/patologíaRESUMEN
Immunotherapy has revolutionized cancer treatment but its efficacy depends on a robust immune response in the tumor. Silencing of the tumor suppressor p53 is common in tumors and can affect the recruitment and activation of different immune cells, leading to immune evasion and poor therapy response. We found that the p53 activating stapled peptide MDM2/MDMX inhibitor Sulanemadlin (ALRN-6924) inhibited p53 wild-type cancer cell growth in vitro and in vivo. In mice carrying p53 wild-type CT26.WT tumors, monotherapy with the PD-1 inhibitor DX400 or Sulanemadlin delayed tumor doubling time by 50% and 37%, respectively, while combination therapy decreased tumor doubling time by 93% leading to an increased median survival time. Sulanemadlin treatment led to increased immunogenicity and combination treatment with PD-1 inhibition resulted in an increased tumor infiltration of lymphocytes. This combination treatment strategy could potentially turn partial responders into responders of immunotherapy, expanding the patient target group for PD-1-targeting immunotherapy.
RESUMEN
Rheumatoid arthritis (RA) is the most common inflammatory joint disease, and early diagnosis is key for effective disease management. CD69 is one of the earliest cell surface markers seen at the surface of activated immune cells, and CD69 is upregulated in synovial tissue in patients with active RA. In this study, we evaluated the performance of a CD69-targeting PET agent, [68Ga]Ga-DOTA-ZCAM241, for early disease detection in a model of inflammatory arthritis. Methods: A model of inflammatory arthritis was induced by transferring splenocytes from KRN T-cell receptor transgenic B6 mice into T-cell-deficient I-Ag7 major histocompatibility complex class II-expressing recipient mice. The mice were examined longitudinally by [68Ga]Ga-DOTA-ZCAM241 PET/CT before and 3, 7, and 12 d after induction of arthritis. Disease progression was monitored by clinical parameters, including measuring body weight and scoring the swelling of the paws. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the paws was analyzed and expressed as SUVmean Tissue biopsy samples were analyzed for CD69 expression by flow cytometry or immunostaining for a histologic correlate. A second group of mice was examined by a nonbinding, size-matched Affibody molecule as the control. Results: Clinical symptoms appeared 5-7 d after induction of arthritis. The uptake of [68Ga]Ga-DOTA-ZCAM241 in the joints was negligible at baseline but increased gradually after disease induction. An elevated PET signal was found on day 3, before the appearance of clinical symptoms. The uptake of [68Ga]Ga-DOTA-ZCAM241 correlated with the clinical score and disease severity. The presence of CD69-positive cells in the joints and lymph nodes was confirmed by flow cytometry and immunostaining. The uptake of the nonbinding tracer that was the negative control also increased gradually with disease progression, although to a lesser extent than with [68Ga]Ga-DOTA-ZCAM241 Conclusion: The uptake of [68Ga]Ga-DOTA-ZCAM241 in the inflamed joints preceded the clinical symptoms in the KRN T-cell transfer model of inflammatory arthritis, in accordance with immunostaining for CD69. [68Ga]Ga-DOTA-ZCAM241 is thus a promising PET imaging marker of activated immune cells in tissue during RA onset.
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Artritis Reumatoide , Tomografía Computarizada por Tomografía de Emisión de Positrones , Humanos , Ratones , Animales , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radioisótopos de Galio , Artritis Reumatoide/metabolismo , Tomografía de Emisión de Positrones , Ratones Transgénicos , Progresión de la EnfermedadRESUMEN
High-throughput drug screening enables the discovery of new anticancer drugs. Although monolayer cell cultures are commonly used for screening, their limited complexity and translational efficiency require alternative models. Three-dimensional cell cultures, such as multicellular tumor spheroids (MCTS), mimic tumor architecture and offer promising opportunities for drug discovery. In this study, we developed a neuroblastoma MCTS model for high-content drug screening. We also aimed to decipher the mechanisms underlying synergistic drug combinations in this disease model. Several agents from different therapeutic categories and with different mechanisms of action were tested alone or in combination with selective inhibition of prostaglandin E2 by pharmacological inhibition of microsomal prostaglandin E synthase-1 (mPGES-1). After a systematic investigation of the sensitivity of individual agents and the effects of pairwise combinations, GFP-transfected MCTS were used in a confirmatory screen to validate the hits. Finally, inhibitory effects on multidrug resistance proteins were examined. In summary, we demonstrate how MCTS-based high-throughput drug screening has the potential to uncover effective drug combinations and provide insights into the mechanism of synergy between an mPGES-1 inhibitor and chemotherapeutic agents.
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Resistencia a Antineoplásicos , Neuroblastoma , Humanos , Prostaglandina-E Sintasas , Esferoides Celulares , Neuroblastoma/tratamiento farmacológico , Descubrimiento de Drogas/métodosRESUMEN
Multiple Sclerosis (MS) is a heterogeneous inflammatory and neurodegenerative disease with an unpredictable course towards progressive disability. Treating progressive MS is challenging due to limited insights into the underlying mechanisms. We examined the molecular changes associated with primary progressive MS (PPMS) using a cross-tissue (blood and post-mortem brain) and multilayered data (genetic, epigenetic, transcriptomic) from independent cohorts. In PPMS, we found hypermethylation of the 1q21.1 locus, controlled by PPMS-specific genetic variations and influencing the expression of proximal genes (CHD1L, PRKAB2) in the brain. Evidence from reporter assay and CRISPR/dCas9 experiments supports a causal link between methylation and expression and correlation network analysis further implicates these genes in PPMS brain processes. Knock-down of CHD1L in human iPSC-derived neurons and knock-out of chd1l in zebrafish led to developmental and functional deficits of neurons. Thus, several lines of evidence suggest a distinct genetic-epigenetic-transcriptional interplay in the 1q21.1 locus potentially contributing to PPMS pathogenesis.
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Encéfalo , Cromosomas Humanos Par 1 , Metilación de ADN , Proteínas de Unión al ADN , Epigénesis Genética , Pez Cebra , Humanos , Pez Cebra/genética , Animales , Metilación de ADN/genética , Cromosomas Humanos Par 1/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , ADN Helicasas/genética , ADN Helicasas/metabolismo , Neuronas/metabolismo , Esclerosis Múltiple Crónica Progresiva/genética , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Femenino , Persona de Mediana Edad , Predisposición Genética a la Enfermedad , AdultoRESUMEN
Apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (SLE). In agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. Still, little is known about how apoptotic cell-derived self-antigens activate autoreactive B cells and where this takes place. In this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. Furthermore, antibodies against scavenger receptors are found before the onset of clinical symptoms in SLE-prone mice, and they are also found in diagnosed SLE patients. Our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. Because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of SLE.
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Apoptosis/inmunología , Autoanticuerpos/inmunología , Tolerancia Inmunológica/inmunología , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/inmunología , Receptores Depuradores/inmunología , Adulto , Animales , Autoantígenos/inmunología , Humanos , Macrófagos/inmunología , Ratones , Ratones Noqueados , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Receptores Depuradores/clasificación , Receptores Depuradores/deficiencia , Receptores Depuradores/genética , Bazo/inmunologíaRESUMEN
Patients with the monogenic disease autoimmune polyendocrine syndrome type I (APSI) develop autoimmunity against multiple endocrine organs and suffer from chronic mucocutaneous candidiasis (CMC), a paradoxical complication with an unknown mechanism. We report here that saliva from APSI patients with CMC is defective in inhibiting growth of Candida albicans in vitro and show reduced levels of a salivary protein identified as cystatin SA1. In contrast, APSI patients without CMC express salivary cystatin SA1 and can inhibit C. albicans to the same extent as healthy controls. We evaluated the anti-fungal activity of cystatin SA1 and found that synthesized full length cystatin SA1 efficiently inhibits growth of C. albicans in vitro. Moreover, APSI patients exhibit salivary IgA autoantibodies recognizing myosin-9, a protein expressed in the salivary glands, thus linking autoimmunity to cystatin SA1 deficiency and CMC. This data suggests an autoimmune mechanism behind CMC in APSI and provides rationale for evaluating cystatin SA1 in antifungal therapy.
Asunto(s)
Candidiasis Mucocutánea Crónica/inmunología , Inhibidores de Crecimiento/metabolismo , Poliendocrinopatías Autoinmunes/inmunología , Cistatinas Salivales/metabolismo , Adulto , Autoanticuerpos/metabolismo , Autoinmunidad , Candidiasis Mucocutánea Crónica/etiología , Candidiasis Mucocutánea Crónica/genética , Femenino , Predisposición Genética a la Enfermedad , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/inmunología , Humanos , Inmunoglobulina A/metabolismo , Masculino , Proteínas Motoras Moleculares/inmunología , Cadenas Pesadas de Miosina/inmunología , Poliendocrinopatías Autoinmunes/complicaciones , Poliendocrinopatías Autoinmunes/genética , Saliva/metabolismo , Cistatinas Salivales/genética , Cistatinas Salivales/inmunología , Adulto JovenRESUMEN
Activation of p53 by small molecule MDM2 inhibitors can induce cell cycle arrest or death in p53 wildtype cancer cells. However, cancer cells exposed to hypoxia can develop resistance to other small molecules, such as chemotherapies, that activate p53. Here, we evaluated whether hypoxia could render cancer cells insensitive to two MDM2 inhibitors with different potencies, nutlin-3a and navtemadlin. Inhibitor efficacy and potency were evaluated under short-term hypoxic conditions in human and mouse cancer cells expressing different p53 genotypes (wild-type, mutant, or null). Treatment of wild-type p53 cancer cells with MDM2 inhibitors reduced cell growth by > 75% in hypoxia through activation of the p53-p21 signaling pathway; no inhibitor-induced growth reduction was observed in hypoxic mutant or null p53 cells except at very high concentrations. The concentration of inhibitors needed to induce the maximal p53 response was not significantly different in hypoxia compared to normoxia. However, inhibitor efficacy varied by species and by cell line, with stronger effects at lower concentrations observed in human cell lines than in mouse cell lines grown as 2D and 3D cultures. Together, these results indicate that MDM2 inhibitors retain efficacy in hypoxia, suggesting they could be useful for targeting acutely hypoxic cancer cells.