RESUMO
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.
Assuntos
Reabsorção Óssea , Maxila , Animais , Camundongos , Maxila/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos Endogâmicos C57BL , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Osteoclastos/metabolismo , Remodelação Óssea , Fosfatidilinositóis/metabolismoRESUMO
The termination of inflammation is governed by endogenous molecules collectively referred to as 'mediators of resolution' of inflammation. There is now strong evidence to suggest that failed resolution may underpin autoimmune and inflammatory diseases and could thus be targeted to decrease inflammation. There are many molecules that have been described as mediators of resolution, and new players are still being continuously discovered. To support the emerging field of 'resolution pharmacology', here we discuss the scientific strategies required to qualify a molecule as a resolution mediator. Systematic definition of the players of resolution, their receptors, and downstream mechanisms remains a necessary knowledge to move the field forward and suggest new targets for the development of novel therapies to treat inflammatory diseases.
Assuntos
Anti-Inflamatórios/metabolismo , Doenças Autoimunes/imunologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/imunologia , Animais , Homeostase , Humanos , Camundongos , FagocitoseRESUMO
The resolution of inflammation is a dynamic process, characterized by the biosynthesis of pro-resolving mediators, including the lipid Lipoxin A4 (LXA4). LXA4 acts on the N-formyl peptide receptor 2 (FPR2/ALX) to mediate anti-inflammatory and pro-resolving effects. In order to exploit the therapeutic potential of endogenous LXA4 in the context of inflammation we have recently developed synthetic LXA4 mimetics (sLXms) including a dimethyl-imidazole-containing FPR2/ALX agonist designated AT-01-KG. Here, we have investigated the effect of treatment with AT-01-KG in established models of articular inflammation. In a model of gout, mice were injected with MSU crystals and treated with AT-01-KG at the peak of inflammatory response. The treatment decreased the number of neutrophils in the knee exudate, an effect which was accompanied by low levels of myeloperoxidase, CXCL1 and IL-1ß in periarticular tissue. AT-01-KG treatment led to reduced tissue damage and hypernociception. The effects of AT-01-KG on neutrophil accumulation were not observed in MSU treated FPR2/3-/-mice. Importantly, AT-01-KG induced resolution of articular inflammation by increasing neutrophil apoptosis and subsequent efficient efferocytosis. In a model of antigen-induced arthritis, AT-01-KG treatment also attenuated inflammatory responses. These data suggest that AT-01-KG may be a potential new therapy for neutrophilic inflammation of the joints.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Supressores da Gota/administração & dosagem , Gota/tratamento farmacológico , Receptores de Formil Peptídeo/agonistas , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Relação Dose-Resposta a Droga , Gota/metabolismo , Gota/patologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Injeções Intra-Articulares/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Formil Peptídeo/metabolismoRESUMO
Resolution failure of exacerbated inflammation triggered by Influenza A virus (IAV) prevents return of pulmonary homeostasis and survival, especially when associated with secondary pneumococcal infection. Therapeutic strategies based on pro-resolving molecules have great potential against acute inflammatory diseases. Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator that acts on its Mas receptor (MasR) to promote resolution of inflammation. We investigated the effects of Ang-(1-7) and the role of MasR in the context of primary IAV infection and secondary pneumococcal infection and evaluated pulmonary inflammation, virus titers and bacteria counts, and pulmonary damage. Therapeutic treatment with Ang-(1-7) decreased neutrophil recruitment, lung injury, viral load and morbidity after a primary IAV infection. Ang-(1-7) induced apoptosis of neutrophils and efferocytosis of these cells by alveolar macrophages, but had no direct effect on IAV replication in vitro. MasR-deficient (MasR-/-) mice were highly susceptible to IAV infection, displaying uncontrolled inflammation, increased viral load and greater lethality rate, as compared to WT animals. Ang-(1-7) was not protective in MasR-/- mice. Interestingly, Ang-(1-7) given during a sublethal dose of IAV infection greatly reduced morbidity associated with a subsequent S. pneumoniae infection, as seen by decrease in the magnitude of neutrophil influx, number of bacteria in the blood leading to a lower lethality. Altogether, these results show that Ang-(1-7) is highly protective against severe primary IAV infection and protects against secondary bacterial infection of the lung. These effects are MasR-dependent. Mediators of resolution of inflammation, such as Ang-(1-7), should be considered for the treatment of pulmonary viral infections.
Assuntos
Angiotensina I/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Infecções Pneumocócicas/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Proteínas Proto-Oncogênicas/imunologia , Receptores Acoplados a Proteínas G/imunologia , Células A549 , Angiotensina I/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Cães , Humanos , Vírus da Influenza A , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Células Madin Darby de Rim Canino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fragmentos de Peptídeos/farmacologia , Peroxidase/imunologia , Fagocitose/efeitos dos fármacos , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/patologia , Pneumonia Viral/imunologia , Pneumonia Viral/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Receptores Acoplados a Proteínas G/genética , Streptococcus pneumoniaeRESUMO
Annexins are well-known Ca2+ phospholipid-binding proteins, which have a wide variety of cellular functions. The role of annexin A1 (AnxA1) in the innate immune system has focused mainly on the anti-inflammatory and proresolving properties through its binding to the formyl-peptide receptor 2 (FPR2)/ALX receptor. However, studies suggesting an intracellular role of AnxA1 are emerging. In this study, we aimed to understand the role of AnxA1 for interleukin (IL)-1ß release in response to activators of the nucleotide-binding domain leucine-rich repeat (NLR) and pyrin domain containing receptor 3 (NLRP3) inflammasome. Using AnxA1 knockout mice, we observed that AnxA1 is required for IL-1ß release in vivo and in vitro. These effects were due to reduction of transcriptional levels of IL-1ß, NLRP3 and caspase-1, a step called NLRP3 priming. Moreover, we demonstrate that AnxA1 co-localize and directly bind to NLRP3, suggesting the role of AnxA1 in inflammasome activation is independent of its anti-inflammatory role via FPR2. Therefore, AnxA1 regulates NLRP3 inflammasome priming and activation in a FPR2-independent manner.
Assuntos
Anexina A1/metabolismo , Inflamassomos/imunologia , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Administração Intranasal , Animais , Cartilagem Articular , Caspase 1/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Gota/induzido quimicamente , Gota/imunologia , Gota/patologia , Humanos , Inflamassomos/metabolismo , Injeções Intra-Articulares , Pulmão/imunologia , Pulmão/patologia , Macrófagos , Masculino , Camundongos , Camundongos Knockout , Cultura Primária de Células , Ligação Proteica/imunologia , Dióxido de Silício/administração & dosagem , Dióxido de Silício/toxicidade , Silicose/imunologia , Silicose/patologia , Transcrição Gênica/imunologia , Ácido Úrico/administração & dosagem , Ácido Úrico/toxicidadeRESUMO
Sphingosine-1-phosphate (S1P) is an important sphingolipid derived from plasma membrane and has a known role in productive phase of inflammation, but its role in neutrophil survival and resolution phase of inflammation is unknown. Here, we investigated the effects of inhibition of S1P receptors and the blockade of S1P synthesis in BALB/c mice and human neutrophils. S1P and S1PR1-3 receptors expression were increased in cells from the pleural cavity stimulated with LPS. Using different antagonists of S1PRs and inhibitors of different steps of the metabolic pathway of S1P production, we show that S1P and its receptors are involved in regulating neutrophil survival and resolution of inflammation in the pleural cavity. Given the role of the S1P-S1PR axis in resolution of inflammation, we sought to identify whether blockade at different levels of the sphingosine-1-phosphate synthesis pathway could affect neutrophil survival in vitro. Inhibitors of the S1P pathway were also able to induce human neutrophil apoptosis. In addition, blockade of S1P synthesis or its receptor facilitated the efferocytosis of apoptotic neutrophil. Taken together, our data demonstrate a fundamental role for S1P in regulating the outcome of inflammatory responses, and position S1P-S1PR axis as a potential target for treatment of neutrophilic inflammation.
Assuntos
Inflamação/imunologia , Lisofosfolipídeos/metabolismo , Neutrófilos/imunologia , Cavidade Pleural/imunologia , Receptores de Esfingosina-1-Fosfato/metabolismo , Esfingosina/análogos & derivados , Animais , Apoptose , Sobrevivência Celular , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ativação de Neutrófilo , Transdução de Sinais , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inibidoresRESUMO
Macrophages are professional phagocytes that display remarkable plasticity, with a range of phenotypes that can be broadly characterized by the M1/M2 dichotomy. Glucocorticoid (GC)-induced leucine zipper (GILZ) is a protein known to mediate anti-inflammatory and some pro-resolving actions, including as neutrophil apoptosis. However, the role of GILZ in key macrophage function is not well understood. Here, we investigated the role of GILZ on macrophage reprogramming and efferocytosis. Using murine bone-marrow-derived macrophages (BMDMs), we found that GILZ was expressed in naive BMDMs and exhibited increased expression in M2-like macrophages (IL4-differentiated). M1-like macrophages (IFN/LPS-differentiated) from GILZ-/- mice showed higher expression of the M1 markers CD86, MHC class II, iNOS, IL-6 and TNF-α, associated with increased levels of phosphorylated STAT1 and lower IL-10 levels, compared to M1-differentiated cells from WT mice. There were no changes in the M2 markers CD206 and arginase-1 in macrophages from GILZ-/- mice differentiated with IL-4, compared to cells from WT animals. Treatment of M1-like macrophages with TAT-GILZ, a cell-permeable GILZ fusion protein, decreased the levels of CD86 and MHC class II in M1-like macrophages without modifying CD206 levels in M2-like macrophages. In line with the in vitro data, increased numbers of M1-like macrophages were found into the pleural cavity of GILZ-/- mice after LPS-injection, compared to WT mice. Moreover, efferocytosis was defective in the context of GILZ deficiency, both in vitro and in vivo. Conversely, treatment of LPS-injected mice with TAT-GILZ promoted inflammation resolution, associated with lower numbers of M1-like macrophages and increased efferocytosis. Collectively, these data indicate that GILZ is a regulator of important macrophage functions, contributing to macrophage reprogramming and efferocytosis, both key steps for the resolution of inflammation.
Assuntos
Apoptose/efeitos dos fármacos , Glucocorticoides/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Animais , Células da Medula Óssea/efeitos dos fármacos , Ensaios de Migração de Leucócitos , Fenômenos Fisiológicos Celulares/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/patologia , Contagem de Leucócitos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Cavidade Pleural/citologiaRESUMO
Inflammation resolution is an active process that functions to restore tissue homeostasis. The participation of the plasminogen (Plg)/plasmin (Pla) system in the productive phase of inflammation is well known, but its involvement in the resolution phase remains unclear. Therefore, we aimed to investigate the potential role of Plg/Pla in key events during the resolution of acute inflammation and its underlying mechanisms. Plg/Pla injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that were primarily macrophages of anti-inflammatory (M2 [F4/80high Gr1- CD11bhigh]) and proresolving (Mres [F4/80med CD11blow]) phenotypes, without changing the number of macrophages with a proinflammatory profile (M1 [F4/80low Gr1+ CD11bmed]). Pleural injection of Plg/Pla also increased M2 markers (CD206 and arginase-1) and secretory products (transforming growth factor ß and interleukin-6) and decreased the expression of inducible nitric oxide synthase (M1 marker). During the resolving phase of lipopolysaccharide (LPS)-induced inflammation when resolving macrophages predominate, we found increased Plg expression and Pla activity, further supporting a link between the Plg/Pla system and key cellular events in resolution. Indeed, Plg or Pla given at the peak of inflammation promoted resolution by decreasing neutrophil numbers and increasing neutrophil apoptosis and efferocytosis in a serine-protease inhibitor-sensitive manner. Next, we confirmed the ability of Plg/Pla to both promote efferocytosis and override the prosurvival effect of LPS via annexin A1. These findings suggest that Plg and Pla regulate several key steps in inflammation resolution, namely, neutrophil apoptosis, macrophage reprogramming, and efferocytosis, which have a major impact on the establishment of an efficient resolution process.
Assuntos
Anexina A1/metabolismo , Reprogramação Celular , Fibrinolisina/metabolismo , Macrófagos/metabolismo , Plasminogênio/metabolismo , Doença Aguda , Animais , Anexina A1/genética , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Fibrinolisina/genética , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/metabolismo , Neutrófilos/patologia , Plasminogênio/genética , Células RAW 264.7RESUMO
Leishmaniases are diseases caused by several Leishmania species. Leishmania (Viannia) braziliensis can cause localized cutaneous leishmaniasis (LCL), which heals spontaneously, or mucosal leishmaniasis (ML), characterized by chronic and intense inflammation and scanty parasitism. Annexin A1 (AnxA1) is a protein involved in modulation and resolution of inflammation through multiple mechanisms. In the present study, the role of AnxA1 was investigated in L. braziliensis-infected BALB/c mice. AnxA1 levels increased at the peak of tissue lesion and parasitism in infected mice. AnxA1 increased also after L. braziliensis infection of BALB/c (wild-type [WT]) bone marrow derived macrophages. Despite a lower parasite intake, parasite burden in bone marrow-derived macrophages from AnxA1-/- mice was similar to WT and associated with an early increase of TNF-α and, later, of IL-10. AnxA1-/- mice controlled tissue parasitism similarly to WT animals, but they developed significantly larger lesions at later stages of infection, with a more pronounced inflammatory infiltrate and increased specific production of IFN-γ, IL-4, and IL-10. AnxA1-/- mice also presented higher phosphorylation levels of ERK-1/2 and p65/RelA (NF-κB) and inducible NO synthase expression, suggesting that AnxA1 may be involved in modulation of inflammation in this model of experimental leishmaniasis. Finally, assessment of AnxA1 levels in sera from patients with LCL or ML revealed that ML patients had higher levels of serum AnxA1 than did LCL patients or control subjects. Collectively, these data indicate that AnxA1 is actively expressed during L. braziliensis infection. In the absence of AnxA1, mice are fully able to control parasite replication, but they present more intense inflammatory responses and delayed ability to resolve their lesion size.
Assuntos
Anexina A1/imunologia , Leishmaniose/imunologia , Macrófagos/imunologia , Adolescente , Adulto , Animais , Western Blotting , Criança , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inflamação/imunologia , Leishmania braziliensis , Leishmaniose/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Adulto JovemRESUMO
Annexin A1 (AnxA1) is a glucocorticoid-regulated protein known for its anti-inflammatory and pro-resolving effects. We have shown previously that the cAMP-enhancing compounds rolipram (ROL; a PDE4 inhibitor) and Bt2cAMP (a cAMP mimetic) drive caspase-dependent resolution of neutrophilic inflammation. In this follow-up study, we investigated whether AnxA1 could be involved in the pro-resolving properties of these compounds using a model of LPS-induced inflammation in BALB/c mice. The treatment with ROL or Bt2cAMP at the peak of inflammation shortened resolution intervals, improved resolution indices, and increased AnxA1 expression. In vitro studies showed that ROL and Bt2cAMP induced AnxA1 expression and phosphorylation, and this effect was prevented by PKA inhibitors, suggesting the involvement of PKA in ROL-induced AnxA1 expression. Akin to these in vitro findings, H89 prevented ROL- and Bt2cAMP-induced resolution of inflammation, and it was associated with decreased levels of intact AnxA1. Moreover, two different strategies to block the AnxA1 pathway (by using N-t-Boc-Met-Leu-Phe, a nonselective AnxA1 receptor antagonist, or by using an anti-AnxA1 neutralizing antiserum) prevented ROL- and Bt2cAMP-induced resolution and neutrophil apoptosis. Likewise, the ability of ROL or Bt2cAMP to induce neutrophil apoptosis was impaired in AnxA-knock-out mice. Finally, in in vitro settings, ROL and Bt2cAMP overrode the survival-inducing effect of LPS in human neutrophils in an AnxA1-dependent manner. Our results show that AnxA1 is at least one of the endogenous determinants mediating the pro-resolving properties of cAMP-elevating agents and cAMP-mimetic drugs.
Assuntos
Anexina A1/agonistas , Bucladesina/uso terapêutico , AMP Cíclico/agonistas , Infiltração de Neutrófilos/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/uso terapêutico , Pleurisia/tratamento farmacológico , Rolipram/uso terapêutico , Animais , Anexina A1/antagonistas & inibidores , Anexina A1/genética , Anexina A1/metabolismo , Apoptose/efeitos dos fármacos , Bucladesina/antagonistas & inibidores , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Inibidores da Fosfodiesterase 4/química , Fosforilação/efeitos dos fármacos , Pleurisia/imunologia , Pleurisia/metabolismo , Pleurisia/patologia , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Células RAW 264.7 , Rolipram/antagonistas & inibidoresRESUMO
Gout is a self-limited inflammatory disease caused by deposition of monosodium urate (MSU) crystals in the joints. Resolution of inflammation is an active process leading to restoration of tissue homeostasis. Here, we studied the role of Annexin A1 (AnxA1), a glucocorticoid-regulated protein that has anti-inflammatory and proresolving actions, in resolution of acute gouty inflammation. Injection of MSU crystals in the knee joint of mice induced inflammation that was associated with expression of AnxA1 during the resolving phase of inflammation. Neutralization of AnxA1 with antiserum or blockade of its receptor with BOC-1 (nonselective) or WRW4 (selective) prevented the spontaneous resolution of gout. There was greater neutrophil infiltration after challenge with MSU crystals in AnxA1 knockout mice (AnxA1-/- ) and delayed resolution associated to decreased neutrophil apoptosis and efferocytosis. Pretreatment of mice with AnxA1-active N-terminal peptide (Ac2-26 ) decreased neutrophil influx, IL-1ß, and CXCL1 production in periarticular joint. Posttreatment with Ac2-26 decreased neutrophil accumulation, IL-1ß, and hypernociception, and improved the articular histopathological score. Importantly, the therapeutic effects of Ac2-26 were associated with increased neutrophils apoptosis and shortened resolution intervals. In conclusion, AnxA1 plays a crucial role in the context of acute gouty inflammation by promoting timely resolution of inflammation.
Assuntos
Anexina A1/metabolismo , Anti-Inflamatórios/uso terapêutico , Gota/tratamento farmacológico , Inflamação/tratamento farmacológico , Articulações/efeitos dos fármacos , Neutrófilos/fisiologia , Peptídeos/uso terapêutico , Animais , Anexina A1/genética , Anexina A1/uso terapêutico , Anticorpos Bloqueadores/administração & dosagem , Apoptose/efeitos dos fármacos , Apoptose/genética , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Gota/induzido quimicamente , Gota/imunologia , Humanos , Inflamação/imunologia , Articulações/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Ácido ÚricoRESUMO
Annexin A1 (AnxA1) is a glucocorticoid-regulated protein endowed with anti-inflammatory and proresolving properties. Intact AnxA1 is a 37-kDa protein that may be cleaved in vivo at the N-terminal region by neutrophil proteases including elastase and proteinase-3, generating the 33-kDa isoform that is largely inactive. In this study, we investigated the dynamics of AnxA1 expression and the effects of synthetic (sivelestat [SIV]; Eglin) and natural (secretory leukocyte protease inhibitor [SLPI]; Elafin) protease inhibitors on the resolution of LPS-induced inflammation. During the settings of LPS inflammation AnxA1 cleavage associated closely with the peak of neutrophil and elastase expression and activity. SLPI expression increased during resolving phase of the pleurisy. Therapeutic treatment of LPS-challenge mice with recombinant human SLPI or Elafin accelerated resolution, an effect associated with increased numbers of apoptotic neutrophils in the pleural exudates, inhibition of elastase, and modulation of the survival-controlling proteins NF-κB and Mcl-1. Similar effects were observed with SIV, which dose-dependently inhibited neutrophil elastase and shortened resolution intervals. Mechanistically, SIV-induced resolution was caspase-dependent, associated to increased levels of intact AnxA1 and decreased expression of NF-κB and Mcl-1. The proresolving effect of antiproteases was also observed in a model of monosodium urate crystals-induced inflammation. SIV skewed macrophages toward resolving phenotypes and enhanced efferocytosis of apoptotic neutrophils. A neutralizing antiserum against AnxA1 and a nonselective antagonist of AnxA1 receptor abolished the accelerated resolution promoted by SIV. Collectively, these results show that elastase inhibition not only inhibits inflammation but actually promotes resolution, and this response is mediated by protection of endogenous intact AnxA1 with ensuing augmentation of neutrophil apoptosis.
Assuntos
Anexina A1/imunologia , Inflamação/imunologia , Inibidores de Proteases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Citometria de Fluxo , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Inflamação/metabolismo , Elastase de Leucócito/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Neutrófilos/imunologia , Inibidores de Proteases/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/farmacologia , Sulfonamidas/farmacologiaRESUMO
Glucocorticoid (GC)-induced leucine zipper (GILZ) has been shown to mediate or mimic several actions of GC. This study assessed the role of GILZ in self-resolving and GC-induced resolution of neutrophilic inflammation induced by LPS in mice. GILZ expression was increased during the resolution phase of LPS-induced pleurisy, especially in macrophages with resolving phenotypes. Pretreating LPS-injected mice with trans-activator of transcription peptide (TAT)-GILZ, a cell-permeable GILZ fusion protein, shortened resolution intervals and improved resolution indices. Therapeutic administration of TAT-GILZ induced inflammation resolution, decreased cytokine levels, and promoted caspase-dependent neutrophil apoptosis. TAT-GILZ also modulated the activation of the survival-controlling proteins ERK1/2, NF-κB and Mcl-1. GILZ deficiency was associated with an early increase of annexin A1 (AnxA1) and did not modify the course of neutrophil influx induced by LPS. Dexamethasone treatment resolved inflammation and induced GILZ expression that was dependent on AnxA1. Dexamethasone-induced resolution was not altered in GILZ(-/-) mice due to compensatory expression and action of AnxA1. Our results show that therapeutic administration of GILZ efficiently induces a proapoptotic program that promotes resolution of neutrophilic inflammation induced by LPS. Alternatively, a lack of endogenous GILZ during the resolution of inflammation is compensated by AnxA1 overexpression.
Assuntos
Inflamação/imunologia , Macrófagos/imunologia , Pleurisia/imunologia , Fatores de Transcrição/imunologia , Animais , Anexina A1/imunologia , Apoptose/imunologia , Western Blotting , Movimento Celular , Modelos Animais de Doenças , Citometria de Fluxo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Pneumococcal pneumonia is a leading cause of mortality worldwide. The inflammatory response to bacteria is necessary to control infection, but it may also contribute to tissue damage. Phosphodiesterase-4 inhibitors, such as rolipram (ROL), effectively reduce inflammation. Here, we examined the impact of ROL in a pneumococcal pneumonia murine model. Mice were infected intranasally with 10(5)-10(6) CFU of Streptococcus pneumoniae, treated with ROL in a prophylactic or therapeutic schedule in combination, or not, with the antibiotic ceftriaxone. Inflammation and bacteria counts were assessed, and ex vivo phagocytosis assays were performed. ROL treatment during S. pneumoniae infection decreased neutrophil recruitment into lungs and airways and reduced lung injury. Prophylactic ROL treatment also decreased cytokine levels in the airways. Although modulation of inflammation by ROL ameliorated pneumonia, bacteria burden was not reduced. On the other hand, antibiotic therapy reduced bacteria without reducing neutrophil infiltration, cytokine level, or lung injury. Combined ROL and ceftriaxone treatment decreased lethality rates and was more efficient in reducing inflammation, by increasing proresolving protein annexin A1 (AnxA1) expression, and bacterial burden by enhancing phagocytosis. Lack of AnxA1 increased inflammation and lethality induced by pneumococcal infection. These data show that immunomodulatory effects of phosphodiesterase-4 inhibitors are useful during severe pneumococcal pneumonia and suggest their potential benefit as adjunctive therapy during infectious diseases.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/enzimologia , Inibidores da Fosfodiesterase 4/uso terapêutico , Pneumonia Pneumocócica/complicações , Pneumonia Pneumocócica/tratamento farmacológico , Pneumonia Pneumocócica/enzimologia , Pneumonia/complicações , Animais , Anexina A1/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ceftriaxona/farmacologia , Ceftriaxona/uso terapêutico , Pulmão/microbiologia , Pulmão/patologia , Lesão Pulmonar/complicações , Lesão Pulmonar/fisiopatologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Pneumonia/tratamento farmacológico , Pneumonia/patologia , Pneumonia/fisiopatologia , Pneumonia Pneumocócica/fisiopatologia , Testes de Função Respiratória , Rolipram/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/fisiologiaRESUMO
The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.
Assuntos
Movimento Celular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Fibrinolisina/imunologia , MAP Quinase Quinase Quinases/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Monócitos/imunologia , Receptor PAR-1/imunologia , Receptores CCR2/imunologia , Animais , Benzoxazinas/farmacologia , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , Cavidade Pleural/imunologia , Receptores CCR2/antagonistas & inibidores , Compostos de Espiro/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Leishmaniases, a group of diseases caused by the species of the protozoan parasite Leishmania, remains a significant public health concern worldwide. Host immune responses play a crucial role in the outcome of Leishmania infections, and several mediators that regulate inflammatory responses are potential targets for therapeutic approaches. Annexin A1 (AnxA1), an endogenous protein endowed with anti-inflammatory and pro-resolving properties, has emerged as a potential player. We have shown that during L. braziliensis infection, deficiency of AnxA1 exacerbates inflammatory responses but does not affect parasite burden. Here, we have investigated the role of AnxA1 in L. amazonensis infection, given the non-healing and progressive lesions characteristic of this infectious model. Infection of AnxA1 KO BALB/c mice resulted in increased lesion size and tissue damage associated with higher parasite burdens and enhanced inflammatory response. Notably, therapeutic application of the AnxA1 peptidomimetic Ac2-26 improves control of parasite replication and increases IL-10 production in vivo and in vitro, in both WT and AnxA1 KO mice. Conversely, administration of WRW4, an inhibitor of FPR2/3, resulted in larger lesions and decreased production of IL-10, suggesting that the effects of AnxA1 during L. amazonensis infection are associated with the engagement of these receptors. Our study illuminates the role of AnxA1 in L. amazonensis infection, demonstrating its impact on the susceptibility phenotype of BALB/c mice. Furthermore, our results indicate that targeting the AnxA1 pathway by using the Ac2-26 peptide could represent a promising alternative for new treatments for leishmaniasis.
Assuntos
Anexina A1 , Leishmania , Leishmaniose , Peptídeos , Animais , Camundongos , Anexina A1/administração & dosagem , Anexina A1/metabolismo , Imunidade , Interleucina-10/metabolismo , Leishmaniose/tratamento farmacológico , Camundongos Endogâmicos BALB C , Peptídeos/administração & dosagemRESUMO
The plasminogen/plasmin (Plg/Pla) system, best known for its classical role in thrombolysis, has been recently highlighted as a regulator of other biological processes in mammals, including key steps involved in the resolution of inflammation. Inflammation resolution is a complex process coordinated by different cellular effectors, notably leukocytes, and active mediators, and is initiated shortly after the inflammatory response begins. Once the inflammatory insult is eliminated, an effective and timely engagement of proresolution programs prevents persistent inflammation, thereby avoiding excessive tissue damage, fibrosis, and the development of autoimmunity. Interestingly, recent studies demonstrate that Plg/Pla and their receptor, plasminogen receptor KT (Plg-RKT), regulate key steps in inflammation resolution. The number of studies investigating the involvement of the Plg/Pla system in these and other aspects of inflammation, including degradation of extracellular matrices, immune cell migration, wound healing, and skeletal growth and maintenance, highlights key roles of the Plg/Pla system during physiological and pathologic conditions. Here, we discuss robust evidence in the literature for the emerging roles of the Plg/Pla system in key steps of inflammation resolution. These findings suggest that dysregulation in Plg production and its activation plays a role in the pathogenesis of inflammatory diseases. Elucidating central mechanisms underlying the role of Plg/Pla in key steps of inflammation resolution either in preclinical models of inflammation or in human inflammatory conditions, can provide a rationale for the development of new pharmacologic interventions to promote resolution of inflammation, and open new pathways for the treatment of thromboinflammatory conditions.
Assuntos
Fibrinolisina , Plasminogênio , Animais , Humanos , Plasminogênio/metabolismo , Fibrinolisina/metabolismo , Macrófagos/metabolismo , Inflamação/metabolismo , Fibrinólise , Mamíferos/metabolismoRESUMO
Macrophages are important effectors of inflammation resolution that contribute to the elimination of pathogens and apoptotic cells and restoration of homeostasis. Pre-clinical studies have evidenced the anti-inflammatory and pro-resolving actions of GILZ (glucocorticoid-induced leucine zipper). Here, we evaluated the role of GILZ on the migration of mononuclear cells under nonphlogistic conditions and Escherichia coli-evoked peritonitis. TAT-GILZ (a cell-permeable GILZ-fusion protein) injection into the pleural cavity of mice induced monocyte/macrophage influx alongside increased CCL2, IL-10 and TGF-ß levels. TAT-GILZ-recruited macrophages showed a regulatory phenotype, exhibiting increased expression of CD206 and YM1. During the resolving phase of E. coli-induced peritonitis, marked by an increased recruitment of mononuclear cells, lower numbers of these cells and CCL2 levels were found in the peritoneal cavity of GILZ-deficient mice (GILZ-/-) when compared to WT. In addition, GILZ-/- showed higher bacterial loads, lower apoptosis/efferocytosis counts and a lower number of macrophages with pro-resolving phenotypes. TAT-GILZ accelerated resolution of E. coli-evoked neutrophilic inflammation, which was associated with increased peritoneal numbers of monocytes/macrophages, enhanced apoptosis/efferocytosis counts and bacterial clearance through phagocytosis. Taken together, we provided evidence that GILZ modulates macrophage migration with a regulatory phenotype, inducing bacterial clearance and accelerating the resolution of peritonitis induced by E. coli.
Assuntos
Infecções por Escherichia coli , Peritonite , Fatores de Transcrição , Animais , Camundongos , Escherichia coli/metabolismo , Infecções por Escherichia coli/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Peritonite/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Joint pain severity in arthritic diseases differs between sexes and is often more pronounced in women. This disparity is thought to stem from biological mechanisms, particularly innate immunity, yet the understanding of sex-specific differences in arthritic pain remains incomplete. This study aims to investigate these disparities using an innate immunity-driven inflammation model induced by intra-articular injections of Streptococcus Cell Wall fragments to mimic both acute and pre-sensitized joint conditions. Nociceptive behavior was evaluated via gait analysis and static weight-bearing, and inflammation was evaluated via joint histology and the synovial gene expression involved in immune response. Although acute inflammation and pain severity were comparable between sexes, distinct associations between synovial inflammatory gene expression and static nociceptive behavior emerged. These associations delineated sex-specific relationships with pain, highlighting differential gene interactions (Il6 versus Cybb on day 1 and Cyba/Gas6 versus Nos2 on day 8) between sexes. In conclusion, our study found that, despite similar pain severity between sexes, the association of inflammatory synovial genes revealed sex-specific differences in the molecular inflammatory mechanisms underlying pain. These findings suggest a path towards more personalized treatment strategies for pain management in arthritis and other inflammatory joint diseases.
Assuntos
Sinovite , Masculino , Humanos , Camundongos , Feminino , Animais , Sinovite/metabolismo , Dor , Inflamação/complicações , Artralgia , Imunidade InataRESUMO
Osteoarthritis (OA) is the most prevalent joint disease, and it is characterized by cartilage degeneration, synovitis, and bone sclerosis, resulting in swelling, stiffness, and joint pain. TAM receptors (Tyro3, Axl, and Mer) play an important role in regulating immune responses, clearing apoptotic cells, and promoting tissue repair. Here, we investigated the anti-inflammatory effects of a TAM receptor ligand, i.e., growth arrest-specific gene 6 (Gas6), in synovial fibroblasts from OA patients. TAM receptor expression was determined in synovial tissue. Soluble Axl (sAxl), a decoy receptor for the ligand Gas6, showed concentrations 4.6 times higher than Gas6 in synovial fluid of OA patients. In OA fibroblast-like synoviocytes (OAFLS) exposed to inflammatory stimuli, the levels of sAxl in the supernatants were increased, while the expression of Gas6 was downregulated. In OAFLS under TLR4 stimulation by LPS (Escherichia coli lipopolysaccharide), the addition of exogenous Gas6 by Gas6-conditioned medium (Gas6-CM) reduced pro-inflammatory markers including IL-6, TNF-α, IL-1ß, CCL2, and CXCL8. Moreover, Gas6-CM downregulated IL-6, CCL2, and IL-1ß in LPS-stimulated OA synovial explants. Pharmacological inhibition of TAM receptors by a pan inhibitor (RU301) or by a selective Axl inhibitor (RU428) similarly abrogated Gas6-CM anti-inflammatory effects. Mechanistically, Gas6 effects were dependent on Axl activation, determined by Axl, STAT1, and STAT3 phosphorylation, and by the downstream induction of the suppressors of the cytokine signaling family (SOCS1 and SOCS3). Taken together, our results showed that Gas6 treatment dampens inflammatory markers of OAFLS and synovial explants derived from OA patients associated with SOCS1/3 production.