RESUMEN

Previous studies have suggested a role of phosphatidylinositol-3-kinase gamma (PI3Kγ) in bone remodeling, but the mechanism remains undefined. Here, we explored the contribution of PI3Kγ in the resorption of maxillary bone and dental roots using models of orthodontic tooth movement (OTM), orthodontic-induced inflammatory root resorption, and rapid maxillary expansion (RME). PI3Kγ-deficient mice (PI3Kγ-/- ), mice with loss of PI3Kγ kinase activity (PI3KγKD/KD ) and C57BL/6 mice treated with a PI3Kγ inhibitor (AS605240) and respective controls were used. The maxillary bones of PI3Kγ-/- , PI3KγKD/KD , and C57BL/6 mice treated with AS605240 showed an improvement of bone quality compared to their controls, resulting in reduction of the OTM and RME in all experimental groups. PI3Kγ-/- mice exhibited increased root volume and decreased odontoclasts counts. Consistently, the pharmacological blockade or genetic deletion of PI3K resulted in increased numbers of osteoblasts and reduction in osteoclasts during OTM. There was an augmented expression of Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (Alp), a reduction of interleukin-6 (Il-6), as well as a lack of responsiveness of receptor activator of nuclear factor kappa-Β (Rank) in PI3Kγ-/- and PI3KγKD/KD mice compared to control mice. The maxillary bones of PI3Kγ-/- animals showed reduced p-Akt expression. In vitro, bone marrow cells treated with AS605240 and cells from PI3Kγ-/- mice exhibited significant augment of osteoblast mineralization and less osteoclast differentiation. The PI3Kγ/Akt axis is pivotal for bone remodeling by providing negative and positive signals for the differentiation of osteoclasts and osteoblasts, respectively.

Asunto(s)

Resorción Ósea , Maxilar , Animales , Ratones , Maxilar/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones Endogámicos C57BL , Resorción Ósea/genética , Resorción Ósea/metabolismo , Osteoclastos/metabolismo , Remodelación Ósea , Fosfatidilinositoles/metabolismo

RESUMEN

We recently demonstrated that clindamycin exhibits activities in acute and chronic models of pain and inflammation. In the present study, we investigated the effects of clindamycin and a clindamycin acetylated derivative (CAD) in models of acute joint inflammation and in a microbiological assay. Joint inflammation was induced in mice by intraarticular (i.a.) injection of zymosan or lipopolysaccharide (LPS). Clindamycin or CAD were administered via the intraperitoneal route 1 h before zymosan or LPS. Paw withdrawal threshold, joint diameter, histological changes, neutrophil recruitment, tumor necrosis factor-α (TNF-α) production and phosphorylation of the IκBα and NF-κB/p65 were evaluated. In vitro assays were used to measure the antibacterial activity of clindamycin and CAD and also their effects on zymosan-induced TNF-α production by RAW264.7 macrophages. Clindamycin exhibited activity against Staphylococcus aureus and Salmonella Typhimurium ATCC® strains at much lower concentrations than CAD. Intraarticular injection of zymosan or LPS induced articular hyperalgesia, edema and neutrophil infiltration in the joints. Zymosan also induced histological changes, NF-κB activation and TNF-α production. Responses induced by zymosan and LPS were inhibited by clindamycin (200 and 400 mg/kg) or CAD (436 mg/kg). Both clindamycin and CAD inhibited in vitro TNF-α production by macrophages. In summary, we provided additional insights of the clindamycin immunomodulatory effects, whose mechanism was associated with NF-κB inhibition and reduced TNF-α production. Such effects were extended to a clindamycin derivative with reduced antibacterial activity, indicating that clindamycin derivatives should be investigated as candidates to drugs that could be useful in the management of inflammatory and painful conditions.

Asunto(s)

Artritis , FN-kappa B , Ratones , Animales , Factor de Necrosis Tumoral alfa/farmacología , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Clindamicina/uso terapéutico , Clindamicina/farmacología , Infiltración Neutrófila , Zimosan , Lipopolisacáridos/farmacología , Inflamación/inducido químicamente , Antibacterianos/farmacología , Edema/inducido químicamente , Edema/tratamiento farmacológico

RESUMEN

Gouty arthritis (GA) is an inflammatory arthritis triggered by the deposition of monosodium urate monohydrate (MSU) crystals, causing pain, inflammation, and joint damage. Several drugs are currently employed to manage acute flares of GA, but they either have limited effectiveness or induce severe adverse reactions. Ouratea spectabilis is traditionally used in Brazil to treat gastric ulcers and rheumatism. The ethanolic extract of O. spectabilis stems (OSpC) and four biflavanones (ouratein A - D) isolated thereof were evaluated in a murine model of GA induced by the injection of MSU crystals. The underlying mechanism of action of ouratein D was investigated in vitro in cell cultures by measurement of IL-1ß levels by ELISA and Western blot analysis. The administration of OSpC (10, 30 or 100 mg/Kg, p. o.) reduced the migration of total inflammatory cells, monocytes, and neutrophils and diminished the levels of IL-1ß and CXCL1 in the synovial tissue. Among the tested compounds, only ouratein D (1 mg/Kg) reduced the migration of the inflammatory cells and it was shown to be active up to 0.01 mg/Kg (equivalent to 0.34 nM/Kg, p. o.). Treatment of pre-stimulated THP-1 cells (differentiated into macrophages) or BMDMs with ouratein D reduced the release of IL-1ß in both macrophage lines. This biflavanone reduced the activation of caspase-1 (showed by the increase in the cleaved form) in supernatants of cultured BMDMs, evidencing its action in modulating the inflammasome pathway. The obtained results demonstrate the anti-gout properties of O. spectabilis and point out ouratein D as the bioactive component of the assayed extract.

Asunto(s)

Artritis Gotosa , Gota , Ochnaceae , Ratones , Animales , Ochnaceae/metabolismo , Gota/inducido químicamente , Gota/metabolismo , Ácido Úrico , Macrófagos/metabolismo , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Artritis Gotosa/inducido químicamente , Artritis Gotosa/tratamiento farmacológico , Artritis Gotosa/metabolismo , Interleucina-1beta/metabolismo

RESUMEN

Gout is a painful form of inflammatory arthritis characterized by the deposition of monosodium urate (MSU) crystals in the joints. The aim of this study was to investigate the effect of peptide P140 on the inflammatory responses in crystal-induced mouse models of gout and cell models including MSU-treated human cells. Injection of MSU crystals into the knee joint of mice induced neutrophil influx and inflammatory hypernociception. Injection of MSU crystals subcutaneously into the hind paw induced edema and increased pro-inflammatory cytokines levels. Treatment with P140 effectively reduced hypernociception, the neutrophil influx, and pro-inflammatory cytokine levels in these experimental models. Furthermore, P140 modulated neutrophils chemotaxis in vitro and increased apoptosis pathways through augmented caspase 3 activity and reduced NFκB phosphorylation. Moreover, P140 increased the production of the pro-resolving mediator annexin A1 and decreased the expression of the autophagy-related ATG5-ATG12 complex and HSPA8 chaperone protein. Overall, these findings suggest that P140 exerts a significant beneficial effect in a neutrophilic inflammation observed in the model of gout that can be of special interest in the design of new therapeutic strategies.

Asunto(s)

Artritis Gotosa , Gota , Ratones , Humanos , Animales , Ácido Úrico , Fosfopéptidos/farmacología , Gota/tratamiento farmacológico , Gota/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Neutrófilos/metabolismo , Modelos Animales de Enfermedad , Artritis Gotosa/tratamiento farmacológico

RESUMEN

BACKGROUND AND PURPOSE: Gouty arthritis is characterized by an intense inflammatory response to monosodium urate crystals (MSU), which induces severe pain. Current therapies are often ineffective in reducing gout-related pain. Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator with anti-inflammatory and analgesic proprieties. In this study, we evaluated the effects and mechanisms of action of RvD1 in an experimental mouse model of gouty arthritis, an aim that was not pursued previously in the literature. EXPERIMENTAL APPROACH: Male mice were treated with RvD1 (intrathecally or intraperitoneally) before or after intraarticular stimulation with MSU. Mechanical hyperalgesia was assessed using an electronic von Frey aesthesiometer. Leukocyte recruitment was determined by knee joint wash cell counting and immunofluorescence. IL-1ß production was measured by ELISA. Phosphorylated NF-kB and apoptosis-associated speck-like protein containing CARD (ASC) were detected by immunofluorescence, and mRNA expression was determined by RT-qPCR. CGRP release was determined by EIA and immunofluorescence. MSU crystal phagocytosis was evaluated by confocal microscopy. KEY RESULTS: RvD1 inhibited MSU-induced mechanical hyperalgesia in a dose- and time-dependent manner by reducing leukocyte recruitment and IL-1ß production in the knee joint. Intrathecal RvD1 reduced the activation of peptidergic neurons and macrophages as well as silenced nociceptor to macrophage communication and macrophage function. CGRP stimulated MSU phagocytosis and IL-1ß production by macrophages. RvD1 downmodulated this phenomenon directly by acting on macrophages, and indirectly by inhibiting CGRP release and CGRP-dependent activation of macrophages. CONCLUSIONS AND IMPLICATIONS: This study reveals a hitherto unknown neuro-immune axis in gouty arthritis that is targeted by RvD1.

Asunto(s)

Artritis Gotosa , Animales , Artritis Gotosa/inducido químicamente , Artritis Gotosa/tratamiento farmacológico , Péptido Relacionado con Gen de Calcitonina , Ácidos Docosahexaenoicos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Inflamación/metabolismo , Activación de Macrófagos , Masculino , Ratones , Neuroinmunomodulación , Neuronas , Nociceptores/metabolismo , Dolor , Ácido Úrico/química , Ácido Úrico/farmacología

RESUMEN

The cardiac circadian clock is responsible for the modulation of different myocardial processes, and its dysregulation has been linked to disease development. How this clock machinery is regulated in the heart remains an open question. Because noradrenaline (NE) can act as a zeitgeber in cardiomyocytes, we tested the hypothesis that adrenergic signaling resets cardiac clock gene expression in vivo. In its anti-phase with Clock and Bmal1, cardiac Per1 abundance increased during the dark phase, concurrent with the rise in heart rate and preceded by an increase in NE levels. Sympathetic denervation altered Bmal1 and Clock amplitude, while Per1 was affected in both amplitude and oscillatory pattern. We next treated mice with a ß-adrenergic receptor (ß-AR) blocker. Strikingly, the ß-AR blockade during the day suppressed the nocturnal increase in Per1 mRNA, without altering Clock or Bmal1. In contrast, activating ß-AR with isoproterenol (ISO) promoted an increase in Per1 expression, demonstrating its responsiveness to adrenergic input. Inhibitors of ERK1/2 and CREB attenuated ISO-induced Per1 expression. Upstream of ERK1/2, PI3Kγ mediated ISO induction of Per1 transcription, while activation of ß2-AR, but not ß1-AR induced increases in ERK1/2 phosphorylation and Per1 expression. Consistent with the ß2-induction of Per1 mRNA, ISO failed to activate ERK1/2 and elevate Per1 in the heart of ß2-AR-/- mice, whereas a ß2-AR antagonist attenuated the nocturnal rise in Per1 expression. Our study established a link between NE/ß2-AR signaling and Per1 oscillation via the PI3Ky-ERK1/2-CREB pathway, providing a new framework for understanding the physiological mechanism involved in resetting cardiac clock genes.

Asunto(s)

Regulación de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Miocardio/metabolismo , Proteínas Circadianas Period/biosíntesis , Receptores Adrenérgicos beta 2/metabolismo , Factores de Transcripción ARNTL/biosíntesis , Factores de Transcripción ARNTL/genética , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Animales , Proteínas CLOCK/biosíntesis , Isoproterenol/farmacología , Masculino , Ratones , Ratones Noqueados , Proteínas Circadianas Period/genética , Receptores Adrenérgicos beta 2/genética

RESUMEN

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD's inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

Asunto(s)

Leucina/efectos de los fármacos , Leucina/genética , Mutación Puntual , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas Inhibidoras de Proteinasas Secretoras/farmacología , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Animales , Artritis , Leucina/química , Leucina/metabolismo , Elastasa de Leucocito/efectos de los fármacos , Ratones , Simulación del Acoplamiento Molecular , Neutrófilos , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas Recombinantes , Relación Estructura-Actividad , Especificidad por Sustrato , Receptor Toll-Like 4/genética , Transcriptoma

RESUMEN

OBJECTIVES: This study aimed to evaluate the involvement of Angiotensin II (Ang II) in joint lesions associated with osteoarthritis (OA) in vitro and in vivo. METHODS: Chondrocyte cultures were obtained from knee joints of neonatal rats and stimulated with Ang II/MIA/ACE inhibitors. In vivo, rats treated or not with the ACE inhibitor captopril, received daily injections of Ang II or sodium monoiodoacetate (MIA) in knee joints for evaluation of cartilage, bone, and synovial lesions. RESULTS: Cultured chondrocytes expressed the mRNA for Ace, Agtr1, Agtr2, and Mas1. Stimulating cells with Ang II reduced chondrocyte viability and metabolism. Accordingly, in vivo Ang II injection into the knees of rats triggered hyperalgesia, joint edema, increased the number of leukocytes in the joint cavity, and induced cartilage lesions associated with OA alterations. In further experiments, Ang II synthesis was prevented with the ACE inhibitor Captopril in the context of MIA-induced OA. Ang II inhibition with captopril improved the OARSI score, induced chondroprotection, and reduced the leukocyte recruitment from synovium after MIA. Additionally, captopril prevented MIA-induced bone resorption, by decreasing the number of osteoclasts and increasing the expression of IL-10 in the bone. In vitro, inhibiting Ang II synthesis decreased MIA-induced chondrocyte death and increased Col2a1 transcription. CONCLUSION: Ang II induces chondrocyte death and joint tissue damages associated with OA and its modulation can be a therapeutic strategy in osteoarthritis.

Asunto(s)

Cartílago Articular , Osteoartritis de la Rodilla , Osteoartritis , Angiotensina II , Animales , Condrocitos , Articulación de la Rodilla , Osteoartritis/tratamiento farmacológico , Osteoartritis de la Rodilla/tratamiento farmacológico , Proto-Oncogenes Mas , Ratas

RESUMEN

OBJECTIVE: To investigate the role of IL-33 in gouty arthritis. MATERIAL: 174 Balb/c (wild-type) and 54 ST2-/- mice were used in this study. In vitro experiments were conducted in bone marrow-derived macrophages (BMDMs). Synovial fluid samples from gouty arthritis (n = 7) and osteoarthritis (n = 8) hospital patients were used to measure IL-33 and sST2 levels. METHODS: Gout was induced by injection of monosodium urate (MSU) crystals in the knee joint of mice. Pain was determined using the electronic von Frey and static weight bearing. Neutrophil recruitment was determined by H&E staining, Rosenfeld staining slides, and MPO activity. ELISA was used for cytokine and sST2 measurement. The priming effect of IL-33 was determined in BMDM. RESULTS: Synovial fluid of gout patients showed higher IL-33 levels and neutrophil counts than osteoarthritis patients. In mice, the absence of ST2 prevented mechanical pain, knee joint edema, neutrophil recruitment to the knee joint, and lowered IL-1ß and superoxide anion levels. In macrophages, IL-33 enhanced the release of IL-1ß and TNF-α, and BMDMs from ST2-/- showed reduced levels of these cytokines after stimulus with MSU crystals. CONCLUSION: IL-33 mediates gout pain and inflammation by boosting macrophages production of cytokines upon MSU crystals stimulus.

Asunto(s)

Artritis Gotosa/patología , Inflamación/inducido químicamente , Interleucina-1beta/metabolismo , Interleucina-33/farmacología , Macrófagos/metabolismo , Dolor/inducido químicamente , Animales , Artritis Gotosa/inducido químicamente , Artritis Gotosa/metabolismo , Femenino , Humanos , Inflamación/psicología , Proteína 1 Similar al Receptor de Interleucina-1/genética , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Persona de Mediana Edad , Infiltración Neutrófila/efectos de los fármacos , Dolor/psicología , Peroxidasa/metabolismo , Superóxidos/metabolismo , Membrana Sinovial/patología , Ácido Úrico

RESUMEN

This study investigates the participation of PI3Kγ in the development of joint inflammation and dysfunction in an experimental model of acute gout in mice. Acute gout was induced by injection of monosodium urate (MSU) crystals into the tibiofemoral joint of mice. The involvement of PI3Kγ was evaluated using a selective inhibitor and mice deficient for PI3Kγ (PI3Kγ-/- ) or with loss of kinase activity. Neutrophils recovered from the inflamed joint were quantified and stained for phosphorylated Akt (pAkt) and production of reactive oxygen species (ROS). The adherence of leukocytes to the joint microvasculature was assessed by intravital microscopy and cleaved caspase-1 by Western blot. Injection of MSU crystals induced massive accumulation of neutrophils expressing phosphorylated Akt. In the absence of PI3Kγ, there was reduction of pAkt expression, chemokine production, and neutrophil recruitment. Genetic or pharmacological inhibition of PI3Kγ reduced the adherence of leukocytes to the joint microvasculature, even in joints with established inflammation. Neutrophils from PI3Kγ-/- mice produced less ROS than wild-type neutrophils. There was decreased joint damage and dysfunction in the absence of PI3Kγ. In addition, in the absence of PI3Kγ activity, there was reduction of cleaved caspase-1 and IL-1ß production in synovial tissue after injection of MSU crystals and leukotriene B4 . Our studies suggest that PI3Kγ is crucial for MSU crystal-induced acute joint inflammation. It is necessary for regulating caspase-1 activation and for mediating neutrophil migration and activation. Drugs that impair PI3Kγ function may be useful to control acute gout inflammation.

Asunto(s)

Artritis Gotosa/enzimología , Artritis Gotosa/inmunología , Caspasa 1/metabolismo , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Infiltración Neutrófila , Enfermedad Aguda , Animales , Adhesión Celular , Movimiento Celular , Fosfatidilinositol 3-Quinasa Clase Ib/deficiencia , Citoplasma/metabolismo , Activación Enzimática , Inflamasomas/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Articulaciones/patología , Leucotrieno B4/metabolismo , Masculino , Ratones Endogámicos C57BL , Microvasos/patología , Neutrófilos/metabolismo , Nocicepción , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Membrana Sinovial/irrigación sanguínea , Ácido Úrico

RESUMEN

The gasotransmitter hydrogen sulfide (H2S) is known to regulate many pathophysiological processes. Preclinical assays have demonstrated that H2S donors exhibit anti-inflammatory and antinociceptive activities, characterized by reduction of inflammatory mediators production, leukocytes recruitment, edema and mechanical allodynia. In the present study, the effects induced by 4-methylbenzenecarbothioamide (4-MBC) in models of pain and inflammation in mice, the mechanisms mediating such effects and the H2S-releasing property of this compound were evaluated. 4-MBC spontaneously released H2S in vitro in the absence of organic thiols. Intraperitoneal (i.p.) administration of 4-MBC (100 or 150 mg/kg) reduced the second phase of the nociceptive response induced by formaldehyde and induced a long lasting inhibitory effect on carrageenan mechanical allodynia. 4-MBC antiallodynic effect was not affected by previous administration of naltrexone or glibenclamide. 4-MBC (50, 100 or 150 mg/kg, i.p.) induced a long lasting inhibitory effect on paw edema induced by carrageenan. The highest dose (150 mg/kg, i.p.) of 4-MBC inhibited tumor necrosis factor-α and CXCL1 production and myeloperoxidase activity induced by carrageenan. Mechanical allodynia and paw edema induced by carrageenan were not inhibited by the 4-MBC oxo analogue (p-toluamide). In summary, 4-MBC, an H2S releasing thiobenzamide, exhibits antinociceptive and anti-inflammatory activities. These activities may be due to reduced cytokine and chemokine production and neutrophil recruitment. The H2S releasing property is likely essential for 4-MBC activity. Our results indicate that 4-MBC may represent a useful pharmacological tool to investigate the biological roles of H2S.

Asunto(s)

Amidas/farmacología , Derivados del Benceno/farmacología , Quimiocina CXCL1/biosíntesis , Sulfuro de Hidrógeno/metabolismo , Dolor/tratamiento farmacológico , Dolor/metabolismo , Tioamidas/farmacología , Factor de Necrosis Tumoral alfa/biosíntesis , Amidas/uso terapéutico , Animales , Derivados del Benceno/química , Derivados del Benceno/uso terapéutico , Modelos Animales de Enfermedad , Edema/tratamiento farmacológico , Hiperalgesia/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Nocicepción/efectos de los fármacos , Tioamidas/química , Tioamidas/uso terapéutico

RESUMEN

CD300a is an inhibitory immunoreceptor expressed in lymphoid and myeloid cells. This study evaluates whether CD300a plays a role in the control of joint inflammation in a model of Ag-induced arthritis (AIA) in mice. CD300a was found to be expressed mostly on neutrophils and its expression was enhanced on neutrophils that migrated to the inflamed synovial cavity. Joint inflammation, as characterized by neutrophil accumulation, was significantly greater in CD300a KO (CD300a-/- ) mice subjected to AIA, as compared to WT mice. This was associated with joint dysfunction, as measured by lower mechanical nociception threshold. There was greater production of the chemokine CXCL1 and the cytokine IL-6 in joints of CD300a-/- mice. In vitro, Mϕs from CD300a-/- mice released higher concentrations of CXCL1 and IL-6 in response to LPS. Splenocytes from immunized CD300a-/- mice produced increased levels of IFN-γ and IL-17 and lower levels of IL-10 when challenged with Ag than cells from WT mice. Neutrophils lacking the CD300a gene had greater chemotactic activity in response to fMLP, CXCL1, and LTB4 than WT neutrophils. In conclusion, these results reveal that the absence of CD300a promotes exacerbation of inflammation in a model of Ag-induced arthritis, suggesting that CD300a is an important receptor for negatively controlling the inflammatory response in this model. Mechanistically, CD300a seems to regulate the activity of various immune cells types involved in the process, including neutrophils, Mϕs, and lymphocytes.

Asunto(s)

Antígenos/efectos adversos , Artritis Experimental/metabolismo , Artritis Experimental/fisiopatología , Inflamación/patología , Receptores Inmunológicos/metabolismo , Animales , Anticuerpos/metabolismo , Artritis Experimental/patología , Quimiotaxis , Citocinas/biosíntesis , Progresión de la Enfermedad , Inflamación/complicaciones , Mediadores de Inflamación/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos BALB C , Infiltración Neutrófila , Neutrófilos/patología , Nocicepción , Dolor/complicaciones , Dolor/patología , Bazo/patología , Membrana Sinovial/patología , Factores de Tiempo

RESUMEN

The purpose of this study was to investigate the role of pentraxin 3 (PTX3), a pivotal component of the innate immune system, in gout. Levels of PTX3 and IL-1ß in human samples were evaluated by ELISA. Development of murine gout was evaluated through the levels of cytokines (PTX3, CXCL1, and IL-1ß) and neutrophil recruitment into the joint cavity. Phagocytosis of monosodium urate (MSU) crystals and caspase-1 activation were determined by flow cytometer. Acute gout patients showed elevated concentration of PTX3 in plasma and synovial fluid as compared with healthy and osteoarthritic subjects. Moreover, there was a positive correlation between intra-articular PTX3 and IL-1ß levels. PTX3 was induced in the periarticular tissue of mice postinjection of MSU crystals. Importantly, Ptx3-deficient mice showed reduced inflammation in response to MSU crystal injection compared with wild-type mice, including reduction of neutrophil recruitment into the joint cavity and IL-1ß and CXCL1 production. Interestingly, addition of PTX3 in vitro enhanced MSU crystal phagocytosis by monocytes and resulted in higher production of IL-1ß by macrophages. This contribution of PTX3 to the phagocytosis of MSU crystals and consequent production of IL-1ß occurred through a mechanism mainly dependent on FcγRIII. Thus, our results suggest that PTX3 acts as a humoral pattern recognition molecule in gout facilitating MSU crystal phagocytosis and contributing to the pathogenesis of gouty arthritis.

Asunto(s)

Artritis Gotosa/inmunología , Proteína C-Reactiva/inmunología , Interleucina-1beta/inmunología , Fagocitosis/inmunología , Componente Amiloide P Sérico/inmunología , Ácido Úrico/inmunología , Animales , Artritis Gotosa/metabolismo , Artritis Gotosa/patología , Proteína C-Reactiva/metabolismo , Humanos , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Componente Amiloide P Sérico/metabolismo , Ácido Úrico/metabolismo

RESUMEN

Gout arthritis is a painful inflammatory disease induced by monosodium urate (MSU) crystals. We evaluate the therapeutic potential of the flavonoid hesperidin methylchalcone (HMC) in a mouse model of gout arthritis induced by intra-articular injection of MSU (100 µg/10 µL). Orally given HMC (3-30 mg/kg, 100 µL) reduced in a dose-dependent manner the MSU-induced hyperalgesia (44%, p < 0.05), edema (54%, p < 0.05), and leukocyte infiltration (70%, p < 0.05). HMC (30 mg/kg) inhibited MSU-induced infiltration of LysM-eGFP+ cells (81%, p < 0.05), synovitis (76%, p < 0.05), and oxidative stress (increased GSH, FRAP, and ABTS by 62, 78, and 73%, respectively; reduced O2- and NO by 89 and 48%, p < 0.05) and modulated cytokine production (reduced IL-1ß, TNF-α, IL-6, and IL-10 by 35, 72, 37, and 46%, respectively, and increased TGF-ß by 90%, p < 0.05). HMC also inhibited MSU-induced NF-κB activation (41%, p < 0.05), gp91phox (66%, p < 0.05) and NLRP3 inflammasome components mRNA expression in vivo (72, 77, 71, and 73% for NLRP3, ASC, pro-caspase-1, and pro-IL-1 ß, respectively, p < 0.05), and induced Nrf2/HO-1 mRNA expression (3.9- and 5.1-fold increase, respectively, p < 0.05). HMC (30, 100, and 300 µM) did not inhibit IL-1ß secretion by macrophages primed by LPS and challenged with MSU (450 µg/mL), demonstrating that the anti-inflammatory effect of HMC in gout arthritis depends on inhibiting NF-κB but not on direct inhibition of inflammasome. The pharmacological effects of HMC indicate its therapeutic potential for the treatment of gout.

Asunto(s)

Artritis Gotosa/tratamiento farmacológico , Chalconas/administración & dosificación , Hesperidina/análogos & derivados , FN-kappa B/metabolismo , Animales , Artritis Gotosa/genética , Artritis Gotosa/metabolismo , Hesperidina/administración & dosificación , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , FN-kappa B/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

The antimicrobial peptide LyeTxI isolated from the venom of the spider Lycosa erythrognatha is a potential model to develop new antibiotics against bacteria and fungi. In this work, we studied a peptide derived from LyeTxI, named LyeTxI-b, and characterized its structural profile and its in vitro and in vivo antimicrobial activities. Compared to LyeTxI, LyeTxI-b has an acetylated N-terminal and a deletion of a His residue, as structural modifications. The secondary structure of LyeTxI-b is a well-defined helical segment, from the second amino acid to the amidated C-terminal, with no clear partition between hydrophobic and hydrophilic faces. Moreover, LyeTxI-b shows a potent antimicrobial activity against Gram-positive and Gram-negative planktonic bacteria, being 10-fold more active than the native peptide against Escherichia coli. LyeTxI-b was also active in an in vivo model of septic arthritis, reducing the number of bacteria load, the migration of immune cells, the level of IL-1ß cytokine and CXCL1 chemokine, as well as preventing cartilage damage. Our results show that LyeTxI-b is a potential therapeutic model for the development of new antibiotics against Gram-positive and Gram-negative bacteria.

RESUMEN

Sepsis is a systemic inflammatory response as a result of uncontrolled infections. Neutrophils are the first cells to reach the primary sites of infection, and chemokines play a key role in recruiting neutrophils. However, in sepsis chemokines could also contribute to neutrophil infiltration to vital organs leading to multiple organ failure. ACKR2 is an atypical chemokine receptor, which can remove and degrade inflammatory CC chemokines. The role of ACK2 in sepsis is unknown. Using a model of cecal ligation and puncture (CLP), we demonstrate here that ACKR2 deficient () mice exhibited a significant reduction in the survival rate compared with similarly treated wild-type (WT) mice. However, neutrophil migration to the peritoneal cavity and bacterial load were similar between WT and ACKR2 mice during CLP. In contrast, ACKR2 mice showed increased neutrophil infiltration and elevated CC chemokine levels in the lung, kidney, and heart compared with the WT mice. In addition, ACKR2 mice also showed more severe lesions in the lung and kidney than those in the WT mice. Consistent with these results, WT mice under nonsevere sepsis (90% survival) had higher expression of ACKR2 in these organs than mice under severe sepsis (no survival). Finally, the lungs from septic patients showed increased number of ACKR2 cells compared with those of nonseptic patients. Our data indicate that ACKR2 may have a protective role during sepsis, and the absence of ACKR2 leads to exacerbated chemokine accumulation, neutrophil infiltration, and damage to vital organs.

Asunto(s)

Insuficiencia Multiorgánica/metabolismo , Infiltración Neutrófila , Neutrófilos/metabolismo , Receptores de Quimiocina/metabolismo , Sepsis/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Insuficiencia Multiorgánica/patología , Neutrófilos/patología , Sepsis/patología

RESUMEN

Septic arthritis is an inflammatory joint disease that is induced by pathogens such as Staphylococcus aureus. Infection of the joint triggers an acute inflammatory response directed by inflammatory mediators including microbial danger signals and cytokines and is accompanied by an influx of leukocytes. The recruitment of these inflammatory cells depends on gradients of chemoattractants including formylated peptides from the infectious agent or dying cells, host-derived leukotrienes, complement proteins and chemokines. Neutrophils are of major importance and play a dual role in the pathogenesis of septic arthritis. On the one hand, these leukocytes are indispensable in the first-line defense to kill invading pathogens in the early stage of disease. However, on the other hand, neutrophils act as mediators of tissue destruction. Since the elimination of inflammatory neutrophils from the site of inflammation is a prerequisite for resolution of the acute inflammatory response, the prolonged stay of these leukocytes at the inflammatory site can lead to irreversible damage to the infected joint, which is known as an important complication in septic arthritis patients. Thus, timely reduction of the recruitment of inflammatory neutrophils to infected joints may be an efficient therapy to reduce tissue damage in septic arthritis.

Asunto(s)

Artritis Infecciosa/terapia , Articulaciones/efectos de los fármacos , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/inmunología , Infecciones Estafilocócicas/terapia , Antibacterianos/uso terapéutico , Artritis Infecciosa/inmunología , Artritis Infecciosa/microbiología , Artritis Infecciosa/cirugía , Artrocentesis/métodos , Artroscopía/métodos , Movimiento Celular/inmunología , Quimiocinas/inmunología , Quimiocinas/metabolismo , Humanos , Inflamación , Articulaciones/inmunología , Articulaciones/microbiología , Articulaciones/cirugía , Leucotrienos/inmunología , Leucotrienos/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/cirugía , Staphylococcus aureus , Succión/métodos

RESUMEN

We investigated the anti-inflammatory and analgesic effects of quercetin in monosodium urate crystals (MSU)-induced gout arthritis, and the sensitivity of quercetin effects to naloxone, an opioid receptor antagonist. Mice were treated with quercetin, and mechanical hyperalgesia was assessed at 1-24 h after MSU injection. In vivo, leukocyte recruitment, cytokine levels, oxidative stress, NFκB activation, and gp91phox and inflammasome components (NLRP3, ASC, Pro-caspase-1, and Pro-IL-1ß) mRNA expression by qPCR were determined in the knee joints at 24 h after MSU injection. Inflammasome activation was determined, in vitro, in lipopolysaccharide-primed macrophages challenged with MSU. Quercetin inhibited MSU-induced mechanical hyperalgesia, leukocyte recruitment, TNFα and IL-1ß production, superoxide anion production, inflammasome activation, decrease of antioxidants levels, NFκB activation, and inflammasome components mRNA expression. Naloxone pre-treatment prevented all the inhibitory effects of quercetin over MSU-induced gout arthritis. These results demonstrate that quercetin exerts analgesic and anti-inflammatory effect in the MSU-induced arthritis in a naloxone-sensitive manner.

RESUMEN

BACKGROUND: Phthalimide analogs have been shown to exhibit anti-inflammatory, analgesic and immunomodulatory activities in different preclinical assays. This study aimed to investigate the potential role of 2-phthalimidethanol (PTD-OH) and 2-phthalimidethyl nitrate (PTD-NO) in a murine model of antigen-induced articular inflammation. METHODS: Articular inflammation was induced by intra-articular injection of methylated bovine serum albumin (mBSA) in the knee joint of immunized male C57BL/6J mice. The animals were pre-treated with PTD-OH or PTD-NO (500mg/kg, per os, - 1h). Nociceptive threshold was measured using an electronic von Frey apparatus. The total number of leukocytes in the synovial cavity was determined. Concentrations of tumor necrosis factor (TNF)-α and CXCL-1 and myeloperoxidase (MPO) activity were determined in periarticular tissue. RESULTS: Both PTD-OH and PTD-NO inhibited at similar extent the mechanical allodynia, neutrophil recruitment to the synovial cavity and periarticular tissue and TNF-α and CXCL-1 production induced by intra-articular challenge with mBSA in immunized mice. CONCLUSIONS: PTD-OH and PTD-NO exhibit a marked activity in a murine model of antigen-induced articular inflammation in immunized animals. These results reinforce the interest in the investigation of phthalimide analogs devoid of the glutarimide ring as candidates to analgesic and anti-inflammatory drugs.

Asunto(s)

Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hiperalgesia/prevención & control , Neutrófilos/efectos de los fármacos , Ftalimidas/farmacología , Analgésicos/farmacología , Animales , Citocinas/genética , Artropatías/inducido químicamente , Artropatías/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Ftalimidas/química , Albúmina Sérica Bovina/inmunología

RESUMEN

The indigenous intestinal microbiota is frequently considered an additional major organ of the human body and exerts profound immunomodulating activities. Germ-free (GF) mice display a significantly different inflammatory responsiveness pattern compared with conventional (CV) mice, and this was dubbed a "hyporesponsive phenotype." Taking into account that the deposition of immune complexes is a major event in acute inflammation and that GF mice have a distinct Ig repertoire and B cell activity, we aimed to evaluate whether this altered Ig repertoire interferes with the inflammatory responsiveness of GF mice. We found that serum transfer from CV naive mice was capable of reversing the inflammatory hyporesponsiveness of GF mice in sterile inflammatory injury induced by intestinal ischemia and reperfusion, as well as in a model of lung infection by Klebsiella pneumoniae Transferring serum from Ig-deficient mice to GF animals did not alter their response to inflammatory insult; however, injecting purified Abs from CV animals restored inflammatory responsiveness in GF mice, suggesting that natural Abs present in serum were responsible for altering GF responsiveness. Mechanistically, injection of serum and Ig from CV mice into GF animals restored IgG deposition, leukocyte influx, NF-κB activation, and proinflammatory gene expression in inflamed tissues and concomitantly downregulated annexin-1 and IL-10 production. Thus, our data show that microbiota-induced natural Abs are pivotal for host inflammatory responsiveness to sterile and infectious insults.

Asunto(s)

Anticuerpos/inmunología , Microbioma Gastrointestinal/inmunología , Vida Libre de Gérmenes , Inflamación/inmunología , Intestinos/inmunología , Animales , Anexinas/inmunología , Anticuerpos/administración & dosificación , Linfocitos B/inmunología , Regulación de la Expresión Génica , Humanos , Interleucina-10/inmunología , Intestinos/microbiología , Intestinos/patología , Isquemia , Infecciones por Klebsiella/inmunología , Infecciones por Klebsiella/microbiología , Klebsiella pneumoniae/inmunología , Pulmón/inmunología , Pulmón/microbiología , Ratones , FN-kappa B/genética