Assuntos
Células Supressoras Mieloides/efeitos dos fármacos , Quinolonas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Tuberculose/tratamento farmacológico , Tuberculose/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Células Supressoras Mieloides/imunologia , Quinolonas/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
Quinoline-3-carboxamides (Q compounds) are immunomodulatory compounds that have shown efficacy both in autoimmune disease and cancer. We have in here investigated the impact of one such compound, paquinimod, on the development of diabetes in the NOD mouse model for type I diabetes (T1D). In cohorts of NOD mice treated with paquinimod between weeks 10 to 20 of age and followed up until 40 weeks of age, we observed dose-dependent reduction in incidence of disease as well as delayed onset of disease. Further, in contrast to untreated controls, the majority of NOD mice treated from 15 weeks of age did not develop diabetes at 30 weeks of age. Importantly, these mice displayed significantly less insulitis, which correlated with selectively reduced number of splenic macrophages and splenic Ly6Chi inflammatory monocytes at end point as compared to untreated controls. Collectively, these results demonstrate that paquinimod treatment can significantly inhibit progression of insulitis to T1D in the NOD mouse. We propose that the effect of paquinimod on disease progression may be related to the reduced number of these myeloid cell populations. Our finding also indicates that this compound could be a candidate for clinical development towards diabetes therapy in humans.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Imunossupressores/uso terapêutico , Quinolinas/uso terapêutico , Animais , Diabetes Mellitus Tipo 1/patologia , Feminino , Glicosúria/prevenção & controle , Imunossupressores/farmacologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NOD , Monócitos/efeitos dos fármacos , Células Mieloides/efeitos dos fármacos , Quinolinas/farmacologiaRESUMO
We show here that increased S100A8 and S100A9 protein expression is induced in spleen of animals with active inflammation or with inoculated tumors. In tumor bearing animals an increased expression was also detected in the lung. To further analyze the induced proteins, we performed chemical cross-linking followed by Western blotting. We observed in protein extracts from spleen that both S100A8/S100A9 heterodimers as well as S100A9 homodimers were formed, both after tumor and inflammatory challenge. The cellular source for S100A9 homodimers were CD11b+GR1+ cells. S100A9 homodimers were also secreted into the extracellular space. Lastly, in the spleen from normal and tumor bearing animals cells expressing relatively higher levels of S100A9 compared to S100A8 could be observed by immunohistochemistry. Taken together, these data show that the biologically potent dimeric form of S100A9 is induced in vivo in situations of tumor burden or inflammatory challenge.
Assuntos
Calgranulina B/metabolismo , Multimerização Proteica , Animais , Calgranulina A/metabolismo , Espaço Extracelular/metabolismo , Inflamação/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Baço/metabolismoRESUMO
Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.
Assuntos
Alarminas/imunologia , Calgranulina A/imunologia , Calgranulina B/imunologia , Alarminas/química , Alarminas/genética , Animais , Artrite/genética , Artrite/imunologia , Artrite/patologia , Sítios de Ligação , Calgranulina A/química , Calgranulina A/genética , Calgranulina B/química , Calgranulina B/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Psoríase/genética , Psoríase/imunologia , Psoríase/patologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
BACKGROUND: Previous work has demonstrated immunomodulatory, anti-tumor, anti-metastatic and anti-angiogenic effects of the small molecule quinoline-3-carboxamide tasquinimod in pre-clinical cancer models. To better understand the anti-tumor effects of tasquinimod in transplantable tumor models, we have evaluated the impact of the compound both on recruitment of myeloid cells to tumor tissue and on tumor-induced myeloid cell expansion as these cells are known to promote tumor development. METHODS: Mice bearing subcutaneous 4 T1 mammary carcinoma tumors were treated with tasquinimod in the drinking water. A BrdU-based flow cytometry assay was utilized to assess the impact of short-term tasquinimod treatment on myeloid cell recruitment to tumors. Additionally, long-term treatment was performed to study the anti-tumor effect of tasquinimod as well as its effects on splenic myeloid cells and their progenitors. Myeloid cell populations were also immune-depleted by in vivo antibody treatment. RESULTS: Short-term tasquinimod treatment did not influence the proliferation of splenic Ly6C(hi) and Ly6G(hi) cells, but instead reduced the influx of Ly6C(hi) cells to the tumor. Treatment with tasquinimod for various periods of time after tumor inoculation revealed that the anti-tumor effect of this compound mainly operated during the first few days of tumor growth. Similar to tasquinimod treatment, antibody-mediated depletion of Ly6C(hi) cells within that same time frame, caused reduced tumor growth, thereby confirming a significant role for these cells in tumor development. Additionally, long-term tasquinimod treatment reduced the splenomegaly and expansion of splenic myeloid cells during a later phase of tumor development. In this phase, tasquinimod normalized the tumor-induced alterations in myeloerythroid progenitor cells in the spleen but had only limited impact on the same populations in the bone marrow. CONCLUSIONS: Our results indicate that tasquinimod treatment reduces tumor growth by operating early after tumor inoculation and that this effect is at least partially caused by reduced recruitment of Ly6C(hi) cells to tumor tissue. Long-term treatment also reduces the number of splenic myeloid cells and myeloerythroid progenitors, but these effects did not influence established rapidly growing tumors.
Assuntos
Inibidores da Angiogênese/farmacologia , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Mieloides/efeitos dos fármacos , Quinolinas/farmacologia , Quinolonas/farmacologia , Administração Oral , Inibidores da Angiogênese/administração & dosagem , Animais , Antígenos Ly/metabolismo , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Células Mieloides/patologia , Células Progenitoras Mieloides/efeitos dos fármacos , Mielopoese/efeitos dos fármacos , Quinolinas/administração & dosagem , Quinolonas/administração & dosagem , Baço/citologia , Baço/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cytosolic Ca2+-binding S100A9 and S100A8 proteins form heterodimers that are primarily expressed in human neutrophils and monocytes. We have recently shown that S100A9 binds to TLR4 in vitro and induces TLR4-dependent NF-κB activation and a pro-inflammatory cytokine response in monocytes. In the present report we have further investigated the S100A9-mediated stimulation of TLR4 in monocytes. Using transmission immunoelectron microscopy, we detected focal binding of S100A9 to monocyte membrane subdomains containing the caveolin-1 protein and TLR4. Furthermore, the S100A9 protein was detected in early endosomes of the stimulated cells, indicating that the protein could be internalized by endocytosis. Although stimulation of monocytes with S100A9 was strictly TLR4-dependent, binding of S100A9 to the plasma membrane and endocytosis of S100A9 was still detectable and coincided with CD14 expression in TLR4-deficient cells. We therefore investigated whether CD14 would be involved in the TLR4-dependent stimulation and could show that the S100A9-induced cytokine response was inhibited both in CD14-deficient cells and in cells exposed to CD14 blocking antibodies. Further, S100A9 was not internalized into CD14-deficient cells suggesting a direct role of CD14 in endocytosis of S100A9. Finally, we could detect satiable binding of S100A9 to CD14 in surface plasmon resonance experiments. Taken together, these results indicate that CD14 is a co-receptor of TLR4 in the S100A9-induced cytokine response.
Assuntos
Calgranulina B/imunologia , Receptores de Lipopolissacarídeos/imunologia , Monócitos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Calgranulina B/genética , Caveolina 1/genética , Caveolina 1/imunologia , Linhagem Celular Tumoral , Membrana Celular/genética , Membrana Celular/imunologia , Citocinas/genética , Citocinas/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Receptores de Lipopolissacarídeos/genética , Camundongos , Camundongos Knockout , Ligação Proteica , Ressonância de Plasmônio de Superfície , Receptor 4 Toll-Like/genéticaRESUMO
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
OBJECTIVE: Levels of S100A8/A9, a proinflammatory and prothrombotic protein complex, are increased in several diseases, and high levels predispose to cardiovascular disease (CVD). Recently, platelet S100A8/A9 synthesis was described in mice and humans in relation to CVD. The aim of this study was to investigate the role of platelet S100A8/A9 in systemic lupus erythematosus (SLE), a disease with markedly increased cardiovascular morbidity, as well as the exact platelet distribution of the S100A8/A9 proteins. METHODS: The occurrence and distribution of platelet S100A8/A9 protein were detected by enzyme-linked immunosorbent assay, electron microscopy, Western blotting, and flow cytometry in healthy controls (n = 79) and in 2 individual cohorts of SLE patients (n = 148 and n = 318, respectively) and related to cardiovascular morbidity. RESULTS: We observed that human platelets expressed S100A8/A9 proteins, and that these were localized in close proximity to intracellular membranes and granules as well as on the cell surface upon activation with physiologic and pathophysiologic stimuli. Interestingly, S100A8/A9 was enriched at sites of membrane interactions, indicating a role of S100A8/A9 in cell-cell communication. S100A8/A9 levels were highly regulated by interferon-α, both in vivo and in vitro. Patients with SLE had increased platelet S100A8/A9 content compared with healthy individuals. Increased levels of platelet S100A8/A9 were associated with CVD, particularly myocardial infarction (odds ratio 4.8, 95% confidence interval 1.5-14.9, P = 0.032 [adjusted for age, sex, and smoking]). CONCLUSION: Platelets contain S100A8/A9 in membrane-enclosed vesicles, enabling rapid cell surface deposition upon activation. Furthermore, platelet S100A8/A9 protein levels were increased in SLE patients, particularly in those with CVD, and may be a future therapeutic target.
Assuntos
Plaquetas , Calgranulina A/sangue , Calgranulina B/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/etiologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/complicações , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Plaquetas/metabolismo , Calgranulina A/biossíntese , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
IgA1 mesangial deposition is the hallmark of IgA nephropathy and Henoch-Schönlein purpura, the onset of which often follows infections. Deposited IgA has been reported as polymeric, J chain associated, and often, hypogalactosylated but with no information concerning the influence of the IgA repertoire or the link between immune stimuli and IgA structure. We explored these issues in the α1KI mouse model, which produces polyclonal human IgA1 prone to mesangial deposition. Compared with mice challenged by a conventional environment, mice in a specific pathogen-free environment had less IgA deposition. However, serum IgA of specific pathogen-free mice showed more galactosylation and much lower polymerization. Notably, wild-type, α1KI, and even J chain-deficient mice showed increased polymeric serum IgA on exposure to pathogens. Strict germfree conditions delayed but did not completely prevent deposition; mice housed in these conditions had very low serum IgA levels and produced essentially monomeric IgA. Finally, comparing monoclonal IgA1 that had different variable regions and mesangial deposition patterns indicated that, independently of glycosylation and polymerization, deposition might also depend on IgA carrying specific variable domains. Together with IgA quantities and constant region post-translational modifications, repertoire changes during immune responses might, thus, modulate IgA propensity to deposition. These IgA features are not associated with circulating immune complexes and C3 deposition and are more pertinent to an initial IgA deposition step preceding overt clinical symptoms in patients.
Assuntos
Mesângio Glomerular/metabolismo , Imunoglobulina A/metabolismo , Animais , Formação de Anticorpos , Feminino , Imunoglobulina A/imunologia , Masculino , Camundongos , Conformação ProteicaRESUMO
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
BACKGROUND: Breast cancer is the most common cancer form among women today. Depending on hormone receptor status, breast cancers are divided into different subtypes with vastly varying prognosis. S100A9 is a calcium-binding protein that is associated with inflammation and expressed not only in myeloid cells but also in some tumours. The role for S100A9 in the malignant cells is not well characterised; however, previous studies have shown that the protein could have important immune-modulating properties. METHODS: Using a human breast cancer cohort consisting of 144 tumour samples and in vitro analysis of human breast cancer cell lines, we investigated the expression and function of S100A9 in human breast cancer. RESULTS: We show that S100A9 expression in breast cancer correlated with the ER(-)PgR(-) breast tumour subtype (P<0.001) and with Ki67 (P=0.024) and was expressed both in the malignant cells and in the tumour-infiltrating anti-inflammatory CD163(+) myeloid cells (P<0.001). Stromal expression of S100A9 also correlated to nodal stage, tumour size and Her2 positivity. Within the ER(-)PgR(-) subgroup, all Her2(+) and EGFR(+) tumours expressed S100A9 in the cytoplasm. Both cytoplasmic staining in the malignant cells as well as stromal S100A9 expression in myeloid cells correlated with a decreased overall survival in breast cancer patients. Furthermore, rS100A9 homodimers induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1ß) in a TLR4- and EGFR-dependent manner in human breast cancer cells in vitro. CONCLUSION: We suggest that S100A9 could be viewed as a novel therapeutic target for patients with ER(-)PgR(-) breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Calgranulina B/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Células MCF-7 , Células Mieloides/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Superfície Celular/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Myeloid-related proteins (MRPs) 8 and 14 are cytosolic proteins secreted from myeloid cells as proinflammatory mediators. Currently, the functional role of circulating extracellular MRP8/14 is unclear. Our present study identifies extracellular MRP8/14 as an autocrine player in the leukocyte adhesion cascade. We show that E-selectin-PSGL-1 interaction during neutrophil rolling triggers Mrp8/14 secretion. Released MRP8/14 in turn activates a TLR4-mediated, Rap1-GTPase-dependent pathway of rapid ß2 integrin activation in neutrophils. This extracellular activation loop reduces leukocyte rolling velocity and stimulates adhesion. Thus, we identify Mrp8/14 and TLR4 as important modulators of the leukocyte recruitment cascade during inflammation in vivo.
Assuntos
Antígenos CD18/metabolismo , Calgranulina A/metabolismo , Calgranulina B/metabolismo , Adesão Celular/fisiologia , Migração e Rolagem de Leucócitos/fisiologia , Neutrófilos/fisiologia , Animais , Antígenos CD18/genética , Calgranulina A/genética , Calgranulina B/genética , Regulação da Expressão Gênica , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Ligação ProteicaRESUMO
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
BACKGROUND: Neutrophilic inflammation often persists for days despite effective antibiotic treatment and contributes to brain damage in bacterial meningitis. We propose here that myeloid-related protein 14 (MRP14), an abundant cytosolic protein in myeloid cells, acts as an endogenous danger signal, driving inflammation and aggravating tissue injury. METHODS: The release pattern of MRP14 was analyzed in human and murine cerebrospinal fluid (CSF), as well as in isolated neutrophils. Its functional role was assessed in a mouse meningitis model, using MRP14-deficient mice. RESULTS: We detected large quantities of MRP14 in CSF specimens from patients and mice with pneumococcal meningitis. Immunohistochemical analyses and a cell-depletion approach indicated neutrophils as the major source of MRP14. In a meningitis model, MRP14-deficient mice showed a better resolution of inflammation during antibiotic therapy, which was accompanied by reduced disease severity. Intrathecal administration of MRP14 before infection reverted the phenotype of MRP14-deficient mice back to wild type. Moreover, intrathecal injection of MRP14 alone was sufficient to induce meningitis in a Toll-like receptor 4 (TLR4)-CXCL2-dependent manner. Finally, treatment with the MRP14 antagonist paquinimod reduced inflammation and disease severity significantly, reaching levels comparable to those achieved after genetic depletion of MRP14. CONCLUSIONS: The present study implicates MRP14 as an essential propagator of inflammation and potential therapeutic target in pneumococcal meningitis.
Assuntos
Calgranulina B/líquido cefalorraquidiano , Meningite Pneumocócica/líquido cefalorraquidiano , Transportadores de Cassetes de Ligação de ATP/líquido cefalorraquidiano , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Estudos de Casos e Controles , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Quimiocina CXCL2/biossíntese , Humanos , Masculino , Meningite Pneumocócica/tratamento farmacológico , Meningite Pneumocócica/imunologia , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
A major barrier for cancer immunotherapy is the presence of suppressive cell populations in patients with cancer, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which contribute to the immunosuppressive microenvironment that promotes tumor growth and metastasis. Tasquinimod is a novel antitumor agent that is currently at an advanced stage of clinical development for treatment of castration-resistant prostate cancer. A target of tasquinimod is the inflammatory protein S100A9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. Here, we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: a tumor vaccine (SurVaxM) for prostate cancer and a tumor-targeted superantigen (TTS) for melanoma. In the combination strategies, tasquinimod inhibited distinct MDSC populations and TAMs of the M2-polarized phenotype (CD206(+)). CD11b(+) myeloid cells isolated from tumors of treated mice expressed lower levels of arginase-1 and higher levels of inducible nitric oxide synthase (iNOS), and were less immunosuppressive ex vivo, which translated into a significantly reduced tumor-promoting capacity in vivo when these cells were coinjected with tumor cells. Tumor-specific CD8(+) T cells were increased markedly in the circulation and in tumors. Furthermore, T-cell effector functions, including cell-mediated cytotoxicity and IFNγ production, were potentiated. Taken together, these data suggest that pharmacologic targeting of suppressive myeloid cells by tasquinimod induces therapeutic benefit and provide the rationale for clinical testing of tasquinimod in combination with cancer immunotherapies.
Assuntos
Antineoplásicos/uso terapêutico , Imunoterapia/métodos , Melanoma Experimental/terapia , Neoplasias da Próstata/terapia , Quinolinas/uso terapêutico , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Castração , Terapia Combinada , Avaliação Pré-Clínica de Medicamentos/métodos , Tolerância Imunológica/efeitos dos fármacos , Masculino , Melanoma Experimental/imunologia , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Transplante de Neoplasias , Neoplasias da Próstata/imunologia , Quinolonas , Subpopulações de Linfócitos T/imunologiaRESUMO
Tasquinimod, an orally active quinoline-3-carboxamide, binds with high affinity to HDAC4 and S100A9 in cancer and infiltrating host cells within compromised tumor microenvironment inhibiting adaptive survival pathways needed for an angiogenic response. Clinical trials document that as low as 0.5-1mg tasquinimod/day is therapeutic against castrate resistant metastatic prostate cancer. Tasquinimod is metabolized via cytochrome P4503A4, but ketoconazole at a dose which completely inhibits CYP3A metabolism does not affect tasquinimod's ability to inhibit endothelial "sprouting" in vitro or anti-cancer efficacy against human prostate cancer xenografts in vivo. Tasquinimod's potency is facilitated by its reversible binding (Kd < 35 µM) to the IIA subdomain of albumin (Sudlow's site I). As blood vessels within the compromised cancer microenvironment are characterized by a higher degree of leakiness than those in normal tissues, this results in an enhanced uptake of tasquinimod bound to albumin in cancer tissue via a tumor specific process known as the "enhanced permeability and retention" (i.e., EPR) effect. Thus, despite plasma levels of < 1 µM, the EPR effect results in intracellular drug concentrations of 2-3 µM, levels several-fold higher than needed for inhibition of endothelial sprouting (IC50 ~ 0.5 µM) or for inhibition of HDAC4 and S100A9 mediated tumor growth.
Assuntos
Inibidores da Angiogênese/farmacocinética , Neoplasias de Próstata Resistentes à Castração/irrigação sanguínea , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Quinolinas/farmacocinética , Albumina Sérica/metabolismo , Microambiente Tumoral , Administração Oral , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/sangue , Animais , Disponibilidade Biológica , Biotransformação , Permeabilidade Capilar , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Orquiectomia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Ligação Proteica , Quinolinas/administração & dosagem , Quinolinas/sangue , Quinolonas , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To elucidate if the density of inflammatory cells expressing S100A9 in malignant and surrounding non-malignant prostate tissues is a prognostic marker for outcome in prostate cancer patients. EXPERIMENTAL DESIGN: Tissue was obtained from 358 men diagnosed with prostate cancer at transurethral resection of the prostate due to obstructive voiding problems, of which 260 were then followed with watchful waiting. Tissue microarrays of both malignant and non-malignant tissues were stained with an antibody against S100A9. The number of stained inflammatory cells was scored and related to clinical outcome and histopathological parameters of known prognostic value. RESULTS: A high number of inflammatory cells expressing S100A9 in both malignant and surrounding non-malignant tissues were associated with significantly shorter cancer-specific survival. This association remained significant when Gleason score and local tumour stage were analysed together with S100A9 in a Cox regression model. Low number of S100A9 positive cells in non-malignant stroma was correlated to significantly longer cancer-specific survival also in patients with Gleason score 8-10 tumours. S100A9 positive cells in tumour stroma were correlated with Gleason score, hyaluronan, platelet-derived growth factor receptor beta (PDGFR-ß), and androgen receptor (inverse correlation) in tumour stroma. S100A9 positive cells in non-malignant stroma correlated with androgen receptor in this tissue compartment (inverse correlation). CONCLUSIONS: A high number of S100A9 positive inflammatory cells in tumour stroma and in non-malignant stroma were associated with shorter cancer-specific survival in prostate cancer patients.
Assuntos
Biomarcadores Tumorais/análise , Calgranulina B/análise , Inflamação/metabolismo , Neoplasias da Próstata/química , Células Estromais/química , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Humanos , Imuno-Histoquímica , Inflamação/patologia , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Neoplasias da Próstata/mortalidade , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Fatores de Risco , Células Estromais/patologia , Fatores de Tempo , Análise Serial de Tecidos , Ressecção Transuretral da Próstata , Resultado do Tratamento , Regulação para Cima , Conduta ExpectanteRESUMO
Quinoline-3-carboxamides (Q-compounds) are currently in clinical development for both autoimmune disease and cancer. We have previously shown that the Q-compound paquinimod (ABR-215757) significantly ameliorates disease symptoms in several mouse models of human inflammatory disease. Considering that recruitment of inflammatory cells into tissue is a common denominator of these models, we have in this report investigated whether paquinimod would interfere with cell accumulation during sterile peritoneal inflammation. To mimic the cell recruitment elicited by tissue injury, we used necrotic cells to induce the acute inflammatory response. We show that per oral treatment with paquinimod significantly reduced the accumulation of Ly6C(hi) inflammatory monocytes and eosinophils, but not neutrophils, in this model, and that this correlated with reduced number of such cells also in the omentum. Treatment also reduced the accumulation of these cell populations at a subcutaneous site of inflammation. In alum-induced inflammation, however, neutrophils were the dominant cell population and paquinimod failed to reduce the accumulation of inflammatory cells. Taken together, our results indicate that paquinimod selectively inhibits cell recruitment during acute sterile inflammation, but that this effect is context-dependent. These data have important implications for the understanding of the mechanism of action of Q-compounds in both pre-clinical and clinical settings.