RESUMEN
Rheumatoid arthritis (RA) is a systemic autoimmune disorder that commonly affects multiple joints of the body. Currently, there is no permanent cure to the disease, but it can be managed with several potent drugs that cause serious side effects on prolonged use. Traditional remedies are considered promising for the treatment of several diseases, particularly chronic conditions, because they have lower side effects compared to synthetic drugs. In folklore, the rhizome of Alpinia calcarata Roscoe (Zingiberaceae) is used as a major ingredient of herbal formulations to treat RA. Phytoconstituents reported in A. calcarata rhizomes are diterpenoids, sesquiterpenoid, flavonoids, phytosterol, and volatile oils. The present study is intended to understand the molecular-level interaction of phytoconstituents present in A. calcarata rhizomes with RA molecular targets using computational approaches. A total of 30 phytoconstituents reported from the plant were used to carry out docking with 36 known targets of RA. Based on the docking results, 4 flavonoids were found to be strongly interacting with the RA targets. Further, molecular dynamics simulation confirmed stable interaction of quercetin with 6 targets (JAK3, SYK, MMP2, TLR8, IRAK1, and JAK1), galangin with 2 targets (IRAK1 and JAK1), and kaempferol (IRAK1) with one target of RA. Moreover, the presence of these three flavonoids was confirmed in the A. calcarata rhizome extract using LC-MS analysis. The computational study suggests that flavonoids present in A. calcarata rhizome may be responsible for RA modulatory activity. Particularly, quercetin and galangin could be potential development candidates for the treatment of RA. Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.
Asunto(s)
Alpinia/química , Artritis Reumatoide/tratamiento farmacológico , Biología Computacional , Flavonoides/farmacología , Fitoquímicos/farmacología , Artritis Reumatoide/metabolismo , Cromatografía Liquida , Flavonoides/análisis , Flavonoides/química , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/efectos de los fármacos , Janus Quinasa 1/química , Janus Quinasa 1/efectos de los fármacos , Janus Quinasa 3/química , Janus Quinasa 3/efectos de los fármacos , Quempferoles/química , Quempferoles/farmacología , Espectrometría de Masas , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fitoquímicos/análisis , Fitoquímicos/química , Extractos Vegetales/química , Quercetina/química , Quercetina/farmacología , Rizoma/química , Quinasa Syk/química , Quinasa Syk/efectos de los fármacos , Receptor Toll-Like 8/química , Receptor Toll-Like 8/efectos de los fármacosRESUMEN
The Toll-like receptor 8 (TLR8) agonist VTX-2337 (motolimod) is an anti-cancer immunotherapeutic agent that is believed to augment natural killer (NK) and dendritic cell (DC) activity. The goal of this work is to examine the role of TLR8 expression/activity in head and neck squamous cell carcinoma (HNSCC) to facilitate the prediction of responders to VTX-2337-based therapy. The prognostic role of TLR8 expression in HNSCC patients was assessed by TCGA and tissue microarray analyses. The anti-tumor effect of VTX-2337 was determined in SCCVII/C3H, mEERL/C57Bl/6 and TUBO-human EGFR/BALB/c syngeneic mouse models. The effect of combined VTX-2337 and cetuximab treatment on tumor growth, survival and immune cell recruitment was assessed. TLR8 expression was associated with CD8+ T cell infiltration and favorable survival outcomes. VTX-2337 delayed tumor growth in all 3 syngeneic mouse models and significantly increased the survival of cetuximab-treated mice. The anti-tumor effects of VTX-2337+ cetuximab were accompanied by increased splenic lymphoid DCs and IFNγ+ CD4+ and tumor-specific CD8+ T cells. Depletion of CD4+ T cells, CD8+ T cells and NK cells were all able to abolish the anti-tumor effect of VTX-2337+ cetuximab. Altogether, VTX-2337 remains promising as an adjuvant for cetuximab-based therapy however patients with high TLR8 expression may be more likely to derive benefit from this drug combination compared to patients with low TLR8 expression.
Asunto(s)
Benzazepinas/farmacología , Cetuximab/farmacología , Linfocitos T/metabolismo , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos/farmacología , Benzazepinas/metabolismo , Biomarcadores Farmacológicos , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Cetuximab/metabolismo , Citocinas/metabolismo , Bases de Datos Genéticas , Femenino , Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Células Asesinas Naturales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Linfocitos T/inmunología , Receptor Toll-Like 8/efectos de los fármacos , Receptor Toll-Like 8/genética , Receptor Toll-Like 8/metabolismoRESUMEN
Type I interferons play a central role in the establishment of an innate immune response against viral infections and tumor cells. Shortly after their discovery in 1957, several groups have looked for small molecules capable of inducing the expression of these cytokines with therapeutic applications in mind. A set of active compounds in mice were identified, but because of their relative inefficiency in humans for reasons not understood at the time, these studies fell into oblivion. In recent years, the characterization of pathogen recognition receptors and the signaling pathways they activate, together with the discovery of plasmacytoid dendritic cells, have revolutionized our understanding of innate immunity. These discoveries and the popularization of high-throughput screening technologies have renewed the interest for small molecules that can induce type I interferons. Proofs about their therapeutic potency in humans are expected very soon.
Asunto(s)
Inductores de Interferón/uso terapéutico , Interferón Tipo I/biosíntesis , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Inductores de Interferón/química , Inductores de Interferón/aislamiento & purificación , Inductores de Interferón/farmacología , Factores Reguladores del Interferón/fisiología , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Ratones , Modelos Moleculares , Estructura Molecular , Nucleósidos/biosíntesis , Producción de Medicamentos sin Interés Comercial , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Conformación Proteica , Receptores de Reconocimiento de Patrones/inmunología , Transducción de Señal , Receptor Toll-Like 8/química , Receptor Toll-Like 8/efectos de los fármacos , Receptores Toll-Like/efectos de los fármacos , Receptores Toll-Like/fisiologíaRESUMEN
BACKGROUND: Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE4) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations. METHODS: In the present study we investigated whether the PDE4 inhibitor piclamilast (10(-6) M) could alter RV or viral mimetic (5 µg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E2 and cAMP production were assayed by ELISA and RV replication was assayed by viral titration. RESULTS: We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines. CONCLUSION: By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE4 inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.
Asunto(s)
Bronquios/virología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/efectos de los fármacos , Citocinas/metabolismo , Miocitos del Músculo Liso/virología , Inhibidores de Fosfodiesterasa 4/farmacología , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Rhinovirus , Receptores Toll-Like/metabolismo , Adolescente , Adulto , Anciano , Aminoquinolinas/farmacología , Benzamidas/farmacología , Bronquios/metabolismo , Bronquios/patología , Células Cultivadas , Comorbilidad , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Humanos , Imiquimod , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Infecciones por Picornaviridae/epidemiología , Infecciones por Picornaviridae/metabolismo , Poli I-C/farmacología , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Enfermedad Pulmonar Obstructiva Crónica/virología , Piridinas/farmacología , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 3/efectos de los fármacos , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/efectos de los fármacos , Receptor Toll-Like 8/agonistas , Receptor Toll-Like 8/efectos de los fármacos , Receptores Toll-Like/efectos de los fármacos , Adulto JovenRESUMEN
Toll-like receptor (TLR)-8 agonists activate adaptive immune responses by inducing robust production of T helper 1-polarizing cytokines, suggesting that TLR8-active compounds may be promising candidate adjuvants. We synthesized and evaluated hitherto unexplored furo[2,3-c]quinolines and regioisomeric furo[3,2-c]quinolines derived via a tandem, one-pot Sonogashira coupling and intramolecular 5-endo-dig cyclization strategy in a panel of primary screens. We observed a pure TLR8-agonistic activity profile in select furo[2,3-c]quinolines, with maximal potency conferred by a C2-butyl group (EC50 = 1.6 µM); shorter, longer, or substituted homologues as well as compounds bearing C1 substitutions were inactive, which was rationalized by docking studies using the recently described crystal structure of human TLR8. The best-in-class compound displayed prominent proinflammatory cytokine induction (including interleukin-12 and interleukin-18), but was bereft of interferon-α inducing properties, confirming its high selectivity for human TLR8.
Asunto(s)
Quinolinas/farmacología , Receptor Toll-Like 8/efectos de los fármacos , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.
Asunto(s)
Oligorribonucleótidos/administración & dosificación , Oligorribonucleótidos/química , Receptor Toll-Like 7/efectos de los fármacos , Receptor Toll-Like 8/efectos de los fármacos , Receptor Toll-Like 9/efectos de los fármacos , Animales , Células Cultivadas , Quimiocinas/efectos de los fármacos , Citocinas/efectos de los fármacos , Femenino , Células HEK293/efectos de los fármacos , Células HEK293/inmunología , Humanos , Inmunidad Innata/efectos de los fármacos , Interferón Tipo I/efectos de los fármacos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Masculino , Ratones , Oligonucleótidos Fosforotioatos/administración & dosificación , Oligonucleótidos Fosforotioatos/química , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
Detection of non-self RNA by TLRs within endosomes and by retinoic acid-inducible gene I (RIG-I)-like helicases in the cytosol is central to mammalian antiviral immunity. In this study, we used pathway-specific agonists and targeted delivery to address RNA immunorecognition in primary human immune cells. Within PBMC, plasmacytoid dendritic cells (pDC) and monocytes were found to be responsible for IFN-alpha production upon immunorecognition of RNA. The mechanisms of RNA recognition in pDC and monocytes were distinct. In pDC, recognition of ssRNA and dsRNA oligonucleotides was TLR7-dependent, whereas a 5' triphosphate moiety (RIG-I ligand activity) had no major contribution to IFN-alpha production. In monocytes, the response to RNA oligonucleotides was mediated by either TLR8 or RIG-I. TLR8 was responsible for IL-12 induction upon endosomal delivery of ssRNA oligonucleotides and RIG-I was responsible for IFN-alpha production upon delivery of 5' triphosphate RNA into the cytosol. In conclusion, the dissection of these pathways by selecting the appropriate structure and delivery of RNA reveals pDC as major producer of IFN-alpha upon TLR-mediated stimulation and monocytes as major producer of IFN-alpha upon RIG-I-mediated stimulation. Furthermore, our results uncover the potential of monocytes to function as major producers of IL-12p70, a key Th1 cytokine classically ascribed to myeloid dendritic cells that cannot be induced by CpG oligonucleotides in the human system.
Asunto(s)
ARN Helicasas DEAD-box/fisiología , Interleucina-12/biosíntesis , Monocitos/inmunología , Oligorribonucleótidos/farmacología , ARN/inmunología , Receptor Toll-Like 7/efectos de los fármacos , Receptor Toll-Like 8/efectos de los fármacos , Células Cultivadas , Proteína 58 DEAD Box , Células Dendríticas/inmunología , Humanos , Interferón-alfa/biosíntesis , Oligorribonucleótidos/inmunología , ARN/farmacología , Receptores InmunológicosRESUMEN
In this chapter we focus on immunorecognition of RNA by two members of the family of Toll-like receptors (TLRs), TLR7, and TLR8. While any long single-stranded RNA is readily recognized by both TLR7 and TLR8, sequencedependent activation of TLR7 and TLR8 becomes more evident when using short RNA oligonucleotides. RNA oligonucleotides containing sequence motifs for TLR7 and TLR8 are termed is RNA (immunostimulatory RNA). Moreover, short doublestranded RNA oligonucleotides as used for siRNA (short interfering RNA) containing such sequences function primarily as ligands for TLR7 but not TLR8. Even in the presence of appropriate sequence motifs, RNA is not detected by TLR7 and TLR8 when certain chemical modifications are present. Both immunological recognition and ignorance are relevant for the development of RNA-based therapeutics, depending on the clinical setting for which they are developed.
Asunto(s)
ARN/fisiología , Receptor Toll-Like 7/fisiología , Receptor Toll-Like 8/fisiología , Animales , Humanos , Inmunización , Ligandos , ARN/inmunología , Transducción de Señal/fisiología , Receptor Toll-Like 7/efectos de los fármacos , Receptor Toll-Like 7/inmunología , Receptor Toll-Like 8/efectos de los fármacos , Receptor Toll-Like 8/inmunologíaRESUMEN
The engagement of Toll-like receptors (TLRs) results in resistance to subsequent challenge with respective ligands in macrophages. Studies have shown that stimulation by ligands for TLR2, TLR4, TLR5 and TLR9 induces this state of hypo-responsiveness (homo-tolerance) towards subsequent stimulation with the same ligands. However, whether homo-tolerance is induced by the ligands of TLR7/8 has not been previously determined. We found that ligands for TLR7/8, namely ss-RNA from HIV and an imidazoquinoline compound, R848, induced macrophage tolerance, as judged by the production of the chemokine MIP-1beta. IRAK-1 phosphorylation was also inhibited in the tolerant cells after subsequent stimulation with R848, although no significant differences were observed in the protein levels of TLR7 between tolerant and non-tolerant cells. These results indicate that macrophage tolerance induced by TLR7/8 ligands is regulated at least at the level of IRAK-1 activation.
Asunto(s)
Tolerancia Inmunológica , Proteínas Inflamatorias de Macrófagos/metabolismo , Macrófagos/inmunología , Quinolinas/farmacología , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismo , Animales , Línea Celular , Quimiocina CCL4 , Ligandos , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/inmunología , Ratones , Receptor Toll-Like 7/efectos de los fármacos , Receptor Toll-Like 8/efectos de los fármacosRESUMEN
Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and play important roles in the innate immune system. While single-stranded viral RNA is the natural ligand of TLR7/TLR8, the imidazoquinoline resiquimod (R-848) is recognized as a potent synthetic agonist of TLR7/TLR8. We investigated the effects of TLR7/8 activation on lipid mediator production in polymorphonuclear leukocytes exposed to R-848. Although R-848 had minimal effects by itself, it strongly enhanced leukotriene B4 formation on subsequent stimulation by fMLP, platelet-activating factor, and the ionophore A23187. R-848 acted via TLR8 but not TLR7 as shown by the lack of effect of the TLR7-specific ligand imiquimod. Priming with R-848 also resulted in enhanced arachidonic acid release and platelet-activating factor formation following fMLP stimulation, as well as enhanced prostaglandin E2 synthesis following the addition of arachidonic acid. Western blot analysis demonstrated that R-848 induced the phosphorylation of the cytosolic phospholipase A2alpha, promoted 5-lipoxygenase translocation and potently stimulated the expression of the type 2 cyclooxygenase. Bafilomycin A1, an inhibitor of endosomal acidification, efficiently inhibited all R-848-induced effects. These studies demonstrate that TLR8 signaling strongly promotes inflammatory lipid mediator biosynthesis and provide novel insights on innate immune response to viral infections.
Asunto(s)
Dinoprostona/biosíntesis , Imidazoles/farmacología , Leucotrieno B4/biosíntesis , Neutrófilos/efectos de los fármacos , Factor de Activación Plaquetaria/biosíntesis , Receptor Toll-Like 7/efectos de los fármacos , Receptor Toll-Like 8/efectos de los fármacos , Humanos , Imidazoles/metabolismo , Mediadores de Inflamación/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Virus ARN/inmunología , Transducción de Señal , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 8/metabolismoRESUMEN
The intestinal innate immune system continually interacts with commensal bacteria, thus oral vaccines should induce extra/alternative activation of DC, potentially through TLR. To examine this we collected intestinal lymph DC (iL-DC) under steady-state conditions and after feeding resiquimod (R-848), a synthetic TLR7/8 ligand, which we showed induces complete emptying of gut DC into lymph. iL-DC are heterogeneous with subset-specific functions. In this study we determined the kinetics of iL-DC subset release, activation and cytokine secretion induced by R-848. We show that L-DC comprise three distinct subsets (CD172ahigh, CD172aint and CD172alow) present with similar frequencies in intestinal but not hepatic lymph. No iL-DC express TLR7 mRNA, and only CD172a+ iL-DC express TLR8. However, after oral R-848 administration, output of all three subsets increases dramatically. CD172ahigh DC release precedes that of CD172alow DC, and the increased frequency of CD25high iL-DC is restricted to the two CD172a+ subsets. After feeding R-848 only CD172ahigh iL-DC secrete IL-6 and IL-12p40. However, CD172aint and CD172ahigh DC secrete similar but markedly lower amounts when stimulated in vitro. These results highlight the importance of in vivo approaches to assess adjuvant effects on DC and give novel insights into the subset-specific effects of an oral TLR ligand on intestinal DC.