RESUMO
BACKGROUND: The interleukin (IL)-1 receptor accessory protein (IL1RAP) is an essential coreceptor required for signalling through the IL-1, IL-33 and IL-36 receptors. Here, we investigate the antifibrotic potential of the combined inhibition of these cytokines by an anti-IL1RAP antibody to provide a scientific background for clinical development in systemic sclerosis (SSc). METHODS: The expression of IL1RAP-associated signalling molecules was determined by data mining of publicly available RNA sequencing (RNAseq) data as well as by imaging mass cytometry. The efficacy of therapeutic dosing of anti-IL1RAP antibodies was determined in three complementary mouse models: sclerodermatous chronic graft-versus-host disease (cGvHD), bleomycin-induced dermal fibrosis model and topoisomerase-I (topo)-induced fibrosis. RESULTS: SSc skin showed upregulation of IL1RAP and IL1RAP-related signalling molecules on mRNA and protein level compared with normal skin. IL-1, IL-33 and IL-36 all regulate distinct gene sets related to different pathophysiological processes in SSc. The responses of human fibroblasts and endothelial cells to IL-1, IL-33 and IL-36 were completely blocked by treatment with an anti-IL1RAP antibody in vitro. Moreover, anti-IL1RAP antibody treatment reduced dermal and pulmonary fibrosis in cGvHD-induced, bleomycin-induced and topoisomerase-induced fibrosis. Importantly, RNAseq analyses revealed effects of IL1RAP inhibition on multiple processes related to inflammation and fibrosis that are also deregulated in human SSc skin. CONCLUSION: This study provides the first evidence for the therapeutic benefits of targeting IL1RAP in SSc. Our findings have high translational potential as the anti-IL1RAP antibody CAN10 has recently entered a phase one clinical trial.
Assuntos
Bleomicina , Modelos Animais de Doenças , Fibrose , Proteína Acessória do Receptor de Interleucina-1 , Interleucina-1 , Interleucina-33 , Fibrose Pulmonar , Escleroderma Sistêmico , Transdução de Sinais , Pele , Animais , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/metabolismo , Interleucina-33/antagonistas & inibidores , Interleucina-33/imunologia , Camundongos , Interleucina-1/antagonistas & inibidores , Proteína Acessória do Receptor de Interleucina-1/antagonistas & inibidores , Pele/patologia , Pele/efeitos dos fármacos , Pele/imunologia , Pele/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/imunologia , Humanos , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , FemininoRESUMO
IL-1α and IL-1ß are both involved in several aspects of tumor biology, including tumor initiation, progression, metastasis, and not least in resistance to various therapies. IL-1α can function as an alarmin to signal cellular stress, and acts to induce downstream events, including production of IL-1ß, to amplify the signal. Both IL-1α and IL-1ß act through the same receptor complex, IL-1R1-IL1RAP, to mediate signal transduction. IL1RAP is expressed on tumor cells and in the tumor microenvironment by for example CAF, macrophages and endothelial cells. The anti-IL1RAP antibody nadunolimab (CAN04) inhibits both IL-1α and IL-1ß signaling and induces ADCC of IL1RAP-expressing tumor cells. As both IL-1α and IL-1ß mediate chemoresistance, the aim of this study was to explore the potential synergy between nadunolimab and chemotherapy. This was performed using the NSCLC PDX model LU2503 and the syngeneic MC38 model, in addition to in vitro cell line experiments. We show that chemotherapy induces expression and release of IL-1α from tumor cells and production of IL-1ß-converting enzyme, ICE, in the tumor stroma. IL-1α is also demonstrated to act on stromal cells to further induce the secretion of IL-1ß, an effect disrupted by nadunolimab. Nadunolimab, and its surrogate antibody, synergize with platinum-based as well as non-platinum-based chemotherapy to induce potent anti-tumor effects, while blockade of only IL-1ß signaling by anti-IL-1ß antibody does not achieve this effect. In conclusion, blockade of IL1RAP with nadunolimab reduces IL-1-induced chemoresistance of tumors.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Células Endoteliais/metabolismo , Interleucina-1beta/metabolismo , Neoplasias/terapia , Transdução de Sinais , Macrófagos/metabolismo , Linhagem Celular , Anticorpos Monoclonais/metabolismo , Caspase 1/metabolismo , Microambiente TumoralRESUMO
BACKGROUND: Interleukin-1 (IL-1) signalling is involved in various protumoural processes including proliferation, immune evasion, metastasis and chemoresistance. CAN04 is a first-in-class monoclonal antibody that binds IL-1 receptor accessory protein (IL1RAP), required for IL-1 signalling. In this first-in-human phase 1 study, we assessed safety, recommended phase 2 dose (RP2D), pharmacokinetics, pharmacodynamics and preliminary anti-tumour activity of CAN04 monotherapy. METHODS: Patients with advanced solid tumours known to express IL1RAP and refractory to standard treatments were enrolled in a dose-escalation study with 5 dose levels (1.0-10.0 mg/kg) of weekly CAN04. RESULTS: Twenty-two patients were enrolled. Most common adverse events were infusion-related reactions (41%), fatigue (32%), constipation (27%), diarrhoea (27%), decreased appetite (23%), nausea (23%) and vomiting (23%). One dose limiting toxicity was reported. No maximum tolerated dose was identified. Pharmacokinetics analyses indicate higher exposures and slower elimination with increasing doses. Decreases in serum IL-6 and CRP were observed in most patients. Twenty-one patients were evaluable for response, 43% had stable disease per immune-related response criteria with no partial/complete responses. CONCLUSIONS: The IL1RAP targeting antibody CAN04 can be safely administered to patients up to 10.0 mg/kg weekly, which was defined as the RP2D. Serum biomarkers supported target engagement and IL-1 pathway inhibition. CLINICAL TRIAL REGISTRATION: NCT03267316.
Assuntos
Antineoplásicos , Neoplasias , Anticorpos Monoclonais/efeitos adversos , Relação Dose-Resposta a Droga , Humanos , Proteína Acessória do Receptor de Interleucina-1/uso terapêutico , Dose Máxima Tolerável , Neoplasias/patologiaRESUMO
AIMS: The interleukin-1 receptor accessory protein (IL1RAP) is a co-receptor required for signalling through the IL-1, IL-33, and IL-36 receptors. Using a novel anti-IL1RAP-blocking antibody, we investigated the role of IL1RAP in atherosclerosis. METHODS AND RESULTS: Single-cell RNA sequencing data from human atherosclerotic plaques revealed the expression of IL1RAP and several IL1RAP-related cytokines and receptors, including IL1B and IL33. Histological analysis showed the presence of IL1RAP in both the plaque and adventitia, and flow cytometry of murine atherosclerotic aortas revealed IL1RAP expression on plaque leucocytes, including neutrophils and macrophages. High-cholesterol diet fed apolipoprotein E-deficient (Apoe-/-) mice were treated with a novel non-depleting IL1RAP-blocking antibody or isotype control for the last 6 weeks of diet. IL1RAP blockade in mice resulted in a 20% reduction in subvalvular plaque size and limited the accumulation of neutrophils and monocytes/macrophages in plaques and of T cells in adventitia, compared with control mice. Indicative of reduced plaque inflammation, the expression of several genes related to leucocyte recruitment, including Cxcl1 and Cxcl2, was reduced in brachiocephalic arteries of anti-IL1RAP-treated mice, and the expression of these chemokines in human plaques was mainly restricted to CD68+ myeloid cells. Furthermore, in vitro studies demonstrated that IL-1, IL-33, and IL-36 induced CXCL1 release from both macrophages and fibroblasts, which could be mitigated by IL1RAP blockade. CONCLUSION: Limiting IL1RAP-dependent cytokine signalling pathways in atherosclerotic mice reduces plaque burden and plaque inflammation, potentially by limiting plaque chemokine production.
Assuntos
Aterosclerose , Inflamação , Proteína Acessória do Receptor de Interleucina-1 , Placa Aterosclerótica , Animais , Feminino , Humanos , Masculino , Camundongos , Anti-Inflamatórios/farmacologia , Aterosclerose/genética , Aterosclerose/patologia , Modelos Animais de Doenças , Inflamação/genética , Inflamação/patologia , Proteína Acessória do Receptor de Interleucina-1/antagonistas & inibidores , Proteína Acessória do Receptor de Interleucina-1/genética , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologia , Transdução de SinaisRESUMO
Interleukin-1 (IL-1)-family cytokines are potent modulators of inflammation, coordinating a vast array of immunological responses across innate and adaptive immune systems. Dysregulated IL-1-family cytokine signaling, however, is involved in a multitude of adverse health effects, such as chronic inflammatory conditions, autoimmune diseases, and cancer. Within the IL-1 family of cytokines, six-IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß, and IL-36γ-require the IL-1 receptor accessory protein (IL-1RAcP) as their shared co-receptor. Common features of cytokine signaling include redundancy of signaling pathways, sharing of cytokines and receptors, pleiotropy of the cytokines themselves, and multifaceted immune responses. Accordingly, targeting multiple cytokines simultaneously is an emerging therapeutic strategy and can provide advantages over targeting a single cytokine pathway. Here, we show that two monoclonal antibodies, CAN10 and 3G5, which target IL-1RAcP for broad blockade of all associated cytokines, do so through distinct mechanisms and provide therapeutic opportunities for the treatment of inflammatory diseases.
Assuntos
Citocinas , Proteína Acessória do Receptor de Interleucina-1 , Transdução de Sinais , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Humanos , Animais , Citocinas/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/imunologia , Camundongos , Inflamação/imunologia , Inflamação/metabolismoRESUMO
BACKGROUND: S100A9 has been shown to be important for the function of so called Myeloid Derived Suppressor Cells (MDSC). Cells with a similar phenotype are also involved in pro-inflammatory processes, and we therefore wanted to investigate the gene expression and function of these cells in animals that were either subjected to chronic inflammation, or inoculated with tumors. METHODS: CD11b(+)Ly6C(++) and Ly6G(+) cells were isolated from spleen, tumor tissue or inflammatory granulomas. S100A9, Arginase 1 and iNOS gene expression in the various CD11b(+) cell populations was analyzed using Q-PCR. The suppressive activity of the CD11b(+) cell populations from different donors was studied in co-culture experiments. RESULTS: S100A9 was shown to be expressed mainly in splenic CD11b(+)Ly6C(+)G(+) cells both at the RNA and protein level. Arginase I and iNOS expression could be detected in both CD11b(+)Ly6C(+)Ly6G(+) and CD11b(+)Ly6C(+)G(-)/C(++)G(-) derived from tumors or a site of chronic inflammation, but was very low in the same cell populations isolated from the spleen. CD11b(+) cells isolated from mice with peritoneal chronic inflammation were able to stimulate T lymphocytes, while CD11b+ cells from mice with peritoneal tumors suppressed T cell growth. CONCLUSION: An identical CD11b(+)Ly6C(++)G(-) cell population appears to have the ability to adopt immune stimulatory or immune suppressive functions dependent on the presence of a local inflammatory or tumor microenvironment. Thus, there is a functional plasticity in the CD11b(+)Ly6C(++)G(-) cell population that cannot be distinguished with the current molecular markers.
Assuntos
Antígenos Ly/metabolismo , Antígeno CD11b/metabolismo , Inflamação/imunologia , Neoplasias/imunologia , Carga Tumoral/imunologia , Animais , Arginase/metabolismo , Calgranulina B/metabolismo , Proliferação de Células , Doença Crônica , Feminino , Inflamação/enzimologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/enzimologia , Neoplasias/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Baço/metabolismo , Baço/patologia , Linfócitos T/citologia , Linfócitos T/imunologia , Terpenos , Microambiente Tumoral/imunologiaRESUMO
Despite more than 25 years of research, the molecular targets of quinoline-3-carboxamides have been elusive although these compounds are currently in Phase II and III development for treatment of autoimmune/inflammatory diseases in humans. Using photoaffinity cross-linking of a radioactively labelled quinoline-3-carboxamide compound, we could determine a direct association between human S100A9 and quinoline-3-carboxamides. This interaction was strictly dependent on both Zn++ and Ca++. We also show that S100A9 in the presence of Zn++ and Ca++ is an efficient ligand of receptor for advanced glycation end products (RAGE) and also an endogenous Toll ligand in that it shows a highly specific interaction with TLR4/MD2. Both these interactions are inhibited by quinoline-3-carboxamides. A clear structure-activity relationship (SAR) emerged with regard to the binding of quinoline-3-carboxamides to S100A9, as well as these compounds potency to inhibit interactions with RAGE or TLR4/MD2. The same SAR was observed when the compound's ability to inhibit acute experimental autoimmune encephalomyelitis in mice in vivo was analysed. Quinoline-3-carboxamides would also inhibit TNFalpha release in a S100A9-dependent model in vivo, as would antibodies raised against the quinoline-3-carboxamide-binding domain of S100A9. Thus, S100A9 appears to be a focal molecule in the control of autoimmune disease via its interactions with proinflammatory mediators. The specific binding of quinoline-3-carboxamides to S100A9 explains the immunomodulatory activity of this class of compounds and defines S100A9 as a novel target for treatment of human autoimmune diseases.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Calgranulina B/metabolismo , Fatores Imunológicos/farmacologia , Inflamação/metabolismo , Quinolinas/metabolismo , Receptores Imunológicos/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Doenças Autoimunes/metabolismo , Cálcio/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/efeitos adversos , Antígeno 96 de Linfócito/metabolismo , Camundongos , Camundongos Knockout , Monócitos/metabolismo , Quinolinas/química , Quinolinas/farmacologia , Quinolinas/uso terapêutico , Receptor para Produtos Finais de Glicação Avançada , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Zinco/metabolismoRESUMO
Interleukin-1 (IL-1) family cytokines are potent mediators of inflammation, acting to coordinate local and systemic immune responses to a wide range of stimuli. Aberrant signaling by IL-1 family cytokine members, however, is linked to myriad inflammatory syndromes, autoimmune conditions and cancers. As such, blocking the inflammatory signals inherent to IL-1 family signaling is an established and expanding therapeutic strategy. While several FDA-approved IL-1 inhibitors exist, including an Fc fusion protein, a neutralizing antibody, and an antagonist cytokine, none specifically targets the co-receptor IL-1 receptor accessory protein (IL-1RAcP). Most IL-1 family cytokines form productive signaling complexes by binding first to their cognate receptors - IL-1RI for IL-1α and IL-1ß; ST2 for IL-33; and IL-36R for IL-36α, IL-36ß and IL-36γ - after which they recruit the shared secondary receptor IL-1RAcP to form a ternary cytokine/receptor/co-receptor complex. Recently, IL-1RAcP was identified as a biomarker for both AML and CML. IL-1RAcP has also been implicated in tumor progression in solid tumors and an anti-IL1RAP antibody (nadunolimab, CAN04) is in phase II clinical studies in pancreatic cancer and non-small cell lung cancer (NCT03267316). As IL-1RAcP is common to all of the abovementioned IL-1 family cytokines, targeting this co-receptor raises the possibility of selective signaling inhibition for different IL-1 family cytokines. Indeed, previous studies of IL-1ß and IL-33 signaling complexes have revealed that these cytokines employ distinct mechanisms of IL-1RAcP recruitment even though their overall cytokine/receptor/co-receptor complexes are structurally similar. Here, using functional, biophysical, and structural analyses, we show that antibodies specific for IL-1RAcP can differentially block signaling by IL-1 family cytokines depending on the distinct IL-1RAcP epitopes that they engage. Our results indicate that targeting a shared cytokine receptor is a viable therapeutic strategy for selective cytokine signaling inhibition.
Assuntos
Anti-Inflamatórios/farmacologia , Anticorpos/farmacologia , Epitopos , Proteína Acessória do Receptor de Interleucina-1/antagonistas & inibidores , Interleucina-1beta/metabolismo , Interleucina-33/metabolismo , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/metabolismo , Anticorpos/imunologia , Anticorpos/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Células HEK293 , Humanos , Proteína Acessória do Receptor de Interleucina-1/imunologia , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Ligação Proteica , Transdução de SinaisRESUMO
Olf-1/early B-cell factor (O/E-1) is a transcription factor important for B-lymphocyte and neuronal gene regulation. Here we report that all three known O/E genes (O/E-1, -2, and -3) are expressed in mouse adipose tissue and are upregulated during adipocyte differentiation. Forced expression of O/E-1 in either the preadipocyte cell line 3T3-L1 or mouse embryonic fibroblasts augmented adipogenesis, and constitutive expression of O/E-1 in uncommitted NIH 3T3 fibroblasts led to initiation of adipocyte differentiation. Furthermore, a dominant negative form of O/E-1 partially suppressed 3T3-L1 adipogenesis, indicating that expression from endogenous O/E target genes is required for 3T3-L1 terminal differentiation. Thus, our data point to the importance of O/E target genes for adipocyte differentiation and suggest a novel role for O/E-1 as an initiator and stimulator of adipogenesis.
Assuntos
Adipócitos/fisiologia , Tecido Adiposo/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Transativadores/genética , Tecido Adiposo/citologia , Animais , Biomarcadores , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/anatomia & histologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Reporter , Camundongos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune dysfunction. A hallmark of SSc is the excessive accumulation of extracellular matrix in the skin and in internal organs. There is a high and unmet medical need for novel therapies in this disease. The pathogenesis of SSc is complex and still poorly understood, but the innate immune system has emerged as an important factor in the disease. SSc patients show increased numbers of macrophages/monocytes in the blood and in the skin compared to healthy individuals and these cells are important sources of profibrotic cytokines and chemokines. Paquinimod belongs to a class of orally active quinoline-3-carboxamide (quinoline) derivatives with immunomodulatory properties and has shown effects in several models of autoimmune/inflammatory disorders. Paquinimod is currently in clinical development for treatment of SSc. The immunomodulatory effects of paquinimod is by targeting the myeloid cell compartment via the S100A9 protein. OBJECTIVE: In this study we investigate whether targeting of myeloid cells by paquinimod can effect disease development in an experimental model of SSc, the tight skin 1 (Tsk-1) mouse model. METHODS: Seven weeks old female B6.Cg-Fbn1(Tsk)/J (Tsk-1) mice were treated with vehicle or paquinimod at the dose of 5 or 25mg/kg/day in the drinking water for 8 weeks. The effect of paquinimod on the level of skin fibrosis and on different subpopulations within the myeloid cell compartment in skin biopsies were evaluated by using histology, immunohistochemisty, a hydroxyproline assay and real-time PCR. Furthermore, the level of IgG in serum from treated animals was also analysed. The statistical analyses were performed using Mann-Whitney nonparametric two tailed rank test. RESULTS: The results show that treatment with paquinimod reduces skin fibrosis measured as reduction of skin thickness and decreased number of myofibroblasts and total hydroxyproline content. The effect on fibrosis was associated with a polarization of macrophages in the skin from a pro-fibrotic M2 to a M1 phenotype. Paquinimod treatment also resulted in a reduced TGFß-response in the skin and an abrogation of the increased auto-antibody production in this SSc model. CONCLUSIONS: Paquinimod reduces skin fibrosis in an experimental model of SSc, and this effect correlates with local and systemic effects on the immune system.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Imunossupressores/uso terapêutico , Macrófagos/efeitos dos fármacos , Quinolinas/uso terapêutico , Escleroderma Sistêmico/tratamento farmacológico , Pele/patologia , Animais , Doenças Autoimunes/imunologia , Calgranulina B/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Imunoglobulina G/sangue , Macrófagos/metabolismo , Camundongos , Escleroderma Sistêmico/patologia , Pele/efeitos dos fármacos , Pele/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Differentiation of multipotent mesenchymal stem cells into lipid-accumulating adipocytes is a physiological process induced by transcription factors in combination with hormonal stimulation. We have used Affymetrix microarrays to compare the adipogenic differentiation pathways of NIH-3T3 fibroblasts induced to undergo in vitro differentiation by ectopic expression of early B cell factor (EBF)-1 or peroxisome proliferator-activated receptor (PPAR)gamma2. These experiments revealed that commitment to the adipogenic pathway in the NIH-3T3 cells was not reflected in gene expression until 4 days after induction of differentiation. Furthermore, gene expression patterns at the earlier time points after stimulation indicated that EBF-1 and PPARgamma2 induced different sets of genes, while the similarities increased upon differentiation, and that several genes linked to adipocyte differentiation were also transiently induced in the vector-transduced cells. These data suggest that the initial activation of genes associated with adipocyte development is independent of commitment to the adipogenic pathway and that EBF-1 and PPARgamma2 induce adipocyte differentiation with comparable kinetics and efficiency.
Assuntos
Adipogenia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , PPAR gama/genética , Transativadores/genética , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/genética , Análise de Variância , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Humanos , Cinética , Ligantes , Camundongos , Células NIH 3T3 , PPAR gama/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/metabolismoRESUMO
The development of a mature B lymphocyte from a bone marrow stem cell is a highly ordered process involving stages with defined features and gene expression patterns. To obtain a deeper understanding of the molecular genetics of this process, we have performed RNA expression analysis of a set of mouse B lineage cell lines representing defined stages of B cell development using Affymetrix microarrays. The cells were grouped based on their previously defined phenotypic features, and a gene expression pattern for each group of cell lines was established. The data indicated that the cell lines representing a defined stage generally presented a high similarity in overall expression profiles. Numerous genes could be identified as expressed with a restricted pattern using dCHIP-based, quantitative comparisons or presence/absence-based, probabilistic state analysis. These experiments provide a model for gene expression during B cell development, and the correctly identified expression patterns of a number of control genes suggest that a series of cell lines can be useful tools in the elucidation of the molecular genetics of a complex differentiation process.
Assuntos
Linfócitos B/citologia , Perfilação da Expressão Gênica , RNA/análise , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Linhagem da Célula , Genes de Imunoglobulinas , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Teoria da ProbabilidadeRESUMO
Upon tissue injury and infection both stressed and dying cells can release proteins that normally reside inside the cells. Some of the released proteins become ligands of various cell surface receptors expressed by local cells and such proteins are denoted as damage associated molecular patterns (DAMPs). Binding of some DAMPs to certain cell surface receptors induces signals emanating in the production of pro-inflammatory cytokines, ultimately leading to an inflammatory response. Our laboratory is interested in the S100A9 protein, a bona fide DAMP protein. This protein normally resides inside monocytes and neutrophils and in these cells it forms heterodimers with the S100A8 protein. The S100A8/A9 heterodimer is released in large amounts during several types of inflammatory disease and is currently used clinically as a biomarker in some diseases. The fact that several different proinflammatory functions have been ascribed to this protein makes it a potential target for the development of small molecule inhibitors. We have developed several such inhibitors, some of which are already in phase III clinical development. This review describes our efforts to investigate the biological functions of the S100A9 protein as well as our ongoing efforts of developing second-generation, more specific, small molecule inhibitors of its pro-inflammatory functions.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antineoplásicos/uso terapêutico , Calgranulina B/metabolismo , Desenho de Fármacos , Mediadores da Inflamação/antagonistas & inibidores , Inflamação/tratamento farmacológico , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Animais , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Ligantes , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway.
Assuntos
Calgranulina A/metabolismo , Calgranulina B/metabolismo , Transporte Biológico , Biotinilação , Western Blotting , Calgranulina A/química , Calgranulina A/genética , Calgranulina B/química , Calgranulina B/genética , Células Cultivadas , Dimerização , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-10/farmacologia , Lisossomos/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Monócitos/citologia , Monócitos/metabolismo , Ressonância de Plasmônio de Superfície , Fator de Necrose Tumoral alfa/farmacologia , Ultracentrifugação , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Tasquinimod (a quinoline-3-carboxyamide) is a small molecule immunotherapy with demonstrated effects on the tumor microenvironment (TME) involving immunomodulation, anti-angiogenesis and inhibition of metastasis. A target molecule of tasquinimod is the inflammatory protein S100A9 which has been shown to affect the accumulation and function of suppressive myeloid cell subsets in tumors. Given the major impact of myeloid cells to the tumor microenvironment, manipulation of this cell compartment is a desirable goal in cancer therapeutics. METHODS: To understand the consequences of tasquinimod treatment on the TME, we evaluated early treatment effects in tumor infiltrating myeloid cells. Cellular phenotypes were studied by flow cytometry while gene expression both in tumor tissue and in isolated CD11b(+) cells or tumor cells were measured by real time-PCR. Effects on angiogenesis were monitored by changes in CD31 levels and by gene expression in tumor tissue. Effects on cytokine levels in tumor tissue and serum were determined by multiplex analysis. RESULTS: The MC38-C215 colon carcinoma tumors showed a substantial infiltration of primarily myeloid cells that were dominated by Ly6C(low)F4/80(+)CD206(+) M2-polarized tumor associated macrophages (TAMs), an immuno-suppressive and pro-angiogenic cell population. Here, we show that tasquinimod treatment induces an anti-tumor effect which is subsequent to a reduction in tumor infiltrating CD206(+) M2 macrophages and a simultaneous increase in M1 macrophages expressing MHC class II and CD86. The tasquinimod-induced changes in TAM polarization were evident within 24 h of exposure, emphasizing the ability of tasquinimod to rapidly reprogram the tumor microenvironment. This change in the tumor associated myeloid compartment preceded an increased IL12-production within the tumor and a decrease in tumor neovascularization. The switch in TAM polarization by tasquinimod was confirmed in the 4T1 breast cancer model where tasquinimod also reduce lung metastasis development. CONCLUSION: Our data show that tasquinimod affects tumor infiltrating myeloid cells early after exposure, leading to a change in phenotype from pro-angiogenic and immunosuppressive M2-like TAMs to pro-inflammatory M1-like macrophages. These changes are consistent with the effects of tasquinimod seen on tumor vascularization, immune suppression and metastasis giving further insights to the anti-tumor mechanism of action of tasquinimod.
RESUMO
Erythroblast transformation-specific domain (ETS) transcription factors regulate some of the critical molecular mechanisms controlling the differentiation of multipotent haematopoietic progenitor cells into effector B-lymphocytes. The SPI-group ETS-protein transcription factors PU.1 and SPI-B play essential and, although coexpressed and binding to similar DNA sequences, unique roles in B-cell differentiation in mice. Mouse SPI-C is an SPI-group ETS protein expressed temporarily during B-cell development and in macrophages. Here we present the genomic organization of the mouse SPI-C gene, and show by rapid amplification of cDNA ends (5'-RACE) analysis that transcription of the mouse SPI-C mRNA starts at a single site producing a single processed transcript. We have also isolated a cDNA clone encoding the human SPI-C homologue, which displays 65% amino acid identity to the murine protein. In addition, we show that the genomic structure of the human and mouse genes are similar, containing a 5' non-coding exon followed by five coding exons. Human SPI-C mRNA is preferentially detected in foetal and adult spleen, lymph nodes and at lower levels in bone marrow and foetal liver. Finally a phylogenetic prediction analysis of SPI-group protein sequences suggest that the SPI-C proteins form a distinct subgroup, with human SPI-C being closest related to the mouse SPI-C protein.
Assuntos
Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Éxons , Genes/genética , Células HeLa , Humanos , Íntrons , Células K562 , Camundongos , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Células Tumorais CultivadasRESUMO
S100A4 and S100A9 proteins have been described as playing roles in the control of tumor growth and metastasis. We show here that a chemical probe, oxyclozanide (OX), selected for inhibiting the interaction between S100A9 and the receptor for advanced glycation end-products (RAGE) interacts with both S100A9 and S100A4. Furthermore, we show that S100A9 and S100A4 interact with RAGE and TLR4; interactions that can be inhibited by OX. Hence, S100A4 and S100A9 display similar functional elements despite their primary sequence diversity. This was further confirmed by showing that S100A4 and S100A9 dimerize both in vitro and in vivo. All of these interactions required levels of Zn++ that are found in the extracellular space but not intracellularly. Interestingly, S100A4 and S100A9 are expressed by distinct CD11b+ subpopulations both in healthy animals and in animals with either inflammatory disease or tumor burden. The functions of S100A9 and S100A4 described in this paper, including heterodimerization, may therefore reflect S100A9 and S100A4 that are released into the extra-cellular milieu.
Assuntos
Calgranulina B/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Linfoma/metabolismo , Sondas Moleculares/metabolismo , Oxiclozanida/metabolismo , Proteínas S100/metabolismo , Animais , Western Blotting , Antígeno CD11b/metabolismo , Calgranulina B/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dimerização , Líquido Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Oxiclozanida/farmacologia , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/química , Receptor 4 Toll-Like/metabolismo , Zinco/metabolismoAssuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/fisiologia , Células Germinativas/metabolismo , Transativadores/metabolismo , Adipócitos/fisiologia , Animais , Linfócitos B/fisiologia , Sistema Nervoso Central/fisiologia , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/genética , Humanos , Modelos Biológicos , Família Multigênica , Filogenia , Transativadores/classificação , Transativadores/genéticaRESUMO
By breeding TRAMP mice with S100A9 knock-out (S100A9(-/-)) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+) S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+) macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/-) and TLR4(-/-), but not in RAGE(-/-) animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFß expression in splenic CD11b(+) cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.
Assuntos
Calgranulina B/metabolismo , Linfoma/metabolismo , Neoplasias da Próstata/metabolismo , Receptor 4 Toll-Like/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno CD11b/metabolismo , Calgranulina B/genética , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Humanos , Linfoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/deficiência , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica/efeitos dos fármacos , Quinolinas/farmacologia , Quinolonas , Baço/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Fator de Crescimento Transformador beta/sangueRESUMO
Ikaros DNA-binding proteins are critical for the development of lymphocytes and other hematopoietic lineages, but it remains unclear how they cooperate with other regulators of signaling and transcription to achieve ordered gene expression during development. Here, we show that Ikaros proteins regulate the pre-BCR component lambda5 in a stage-specific manner. In pre-BI cells, Ikaros modulated lambda5 expression in competition with the transcriptional activator EBF. This required Ikaros binding to the Igll1 (lambda5) promoter and was abolished either by mutation of the Ikaros DNA-binding domain or by deletion of a single Ikaros site from the Igll1 promoter. At the transition from the pre-BI to pre-BII stage, the expression of the Ikaros family member Aiolos was upregulated and required for the efficient silencing of Igll1. Aiolos expression was controlled by pre-BCR signals via the adaptor protein SLP-65. Thus, pre-BCR signaling regulates Aiolos and the silencing of Igll1 via a developmental-stage-specific feedback loop.