Blocking the HGF-MET pathway induces resolution of neutrophilic inflammation by promoting neutrophil apoptosis and efferocytosis.

Inflammation resolution is an active process that involves cellular events such as apoptosis and efferocytosis, which are key steps in the restoration of tissue homeostasis. Hepatocyte growth factor (HGF) is a growth factor mostly produced by mesenchymal-origin cells and has been described to act via MET receptor tyrosine kinase. The HGF/MET axis is essential for determining the progression and severity of inflammatory and immune-mediated disorders. Here, we investigated the effect of blocking the HGF/MET signalling pathway by PF-04217903 on the resolution of established models of neutrophilic inflammation. In a self-resolving model of gout induced by MSU crystals, HGF expression on periarticular tissue peaked at 12 h, the same time point that neutrophils reach their maximal accumulation in the joints. The HGF/MET axis was activated in this model, as demonstrated by increased levels of MET phosphorylation in neutrophils (Ly6G+ cells). In addition, the number of neutrophils was reduced in the knee exudate after PF-04217903 treatment, an effect accompanied by increased neutrophil apoptosis and efferocytosis and enhanced expression of Annexin A1, a key molecule for inflammation resolution. Reduced MPO activity, IL-1ß and CXCL1 levels were also observed in periarticular tissue. Importantly, PF-04217903 reduced the histopathological score and hypernociceptive response. Similar findings were obtained in LPS-induced neutrophilic pleurisy. In human neutrophils, the combined use of LPS and HGF increased MET phosphorylation and provided a prosurvival signal, whereas blocking MET with PF-04217903 induced caspase-dependent neutrophil apoptosis. Taken together, these data demonstrate that blocking HGF/MET signalling may be a potential therapeutic strategy for inducing the resolution of neutrophilic inflammatory responses.

Biochanin A as a modulator of the inflammatory response: An updated overview and therapeutic potential.

Uncontrolled inflammation and failure to resolve the inflammatory response are crucial factors involved in the progress of inflammatory diseases. Current therapeutic strategies aimed at controlling excessive inflammation are effective in some cases, though they may be accompanied by severe side effects, such as immunosuppression. Phytochemicals as a therapeutic alternative can have a fundamental impact on the different stages of inflammation and its resolution. Biochanin A (BCA) is an isoflavone known for its wide range of pharmacological properties, especially its marked anti-inflammatory effects. Recent studies have provided evidence of BCA's abilities to activate events essential for resolving inflammation. In this review, we summarize the most recent findings from pre-clinical studies of the pharmacological effects of BCA on the complex signaling network associated with the onset and resolution of inflammation and BCA's potential protective functionality in several models of inflammatory diseases, such as arthritis, pulmonary disease, neuroinflammation, and metabolic disease.

Angiotensin-(1-7)/MasR axis promotes migration of monocytes/macrophages with a regulatory phenotype to perform phagocytosis and efferocytosis.

Nonphlogistic migration of macrophages contributes to the clearance of pathogens and apoptotic cells, a critical step for the resolution of inflammation and return to homeostasis. Angiotensin-(1-7) [Ang-(1-7)] is a heptapeptide of the renin-angiotensin system that acts through Mas receptor (MasR). Ang-(1-7) has recently emerged as a novel proresolving mediator, yet Ang-(1-7) resolution mechanisms are not fully determined. Herein, Ang-(1-7) stimulated migration of human and murine monocytes/macrophages in a MasR-, CCR2-, and MEK/ERK1/2-dependent manner. Pleural injection of Ang-(1-7) promoted nonphlogistic mononuclear cell influx alongside increased levels of CCL2, IL-10, and macrophage polarization toward a regulatory phenotype. Ang-(1-7) induction of CCL2 and mononuclear cell migration was also dependent on MasR and MEK/ERK. Of note, MasR was upregulated during the resolution phase of inflammation, and its pharmacological inhibition or genetic deficiency impaired mononuclear cell recruitment during self-resolving models of LPS pleurisy and E. coli peritonitis. Inhibition/absence of MasR was associated with reduced CCL2 levels, impaired phagocytosis of bacteria, efferocytosis, and delayed resolution of inflammation. In summary, we have uncovered a potentially novel proresolving feature of Ang-(1-7), namely the recruitment of mononuclear cells favoring efferocytosis, phagocytosis, and resolution of inflammation. Mechanistically, cell migration was dependent on MasR, CCR2, and the MEK/ERK pathway.

Biochanin A Regulates Key Steps of Inflammation Resolution in a Model of Antigen-Induced Arthritis via GPR30/PKA-Dependent Mechanism.

Biochanin A (BCA) is a natural organic compound of the class of phytochemicals known as flavonoids and isoflavone subclass predominantly found in red clover (Trifolium pratense). It has anti-inflammatory activity and some pro-resolving actions, such as neutrophil apoptosis. However, the effect of BCA in the resolution of inflammation is still poorly understood. In this study, we investigated the effects of BCA on the neutrophilic inflammatory response and its resolution in a model of antigen-induced arthritis. Male wild-type BALB/c mice were treated with BCA at the peak of the inflammatory process (12 h). BCA decreased the accumulation of migrated neutrophils, and this effect was associated with reduction of myeloperoxidase activity, IL-1ß and CXCL1 levels, and the histological score in periarticular tissues. Joint dysfunction, as seen by mechanical hypernociception, was improved by treatment with BCA. The resolution interval (Ri) was also quantified, defining profiles of acute inflammatory parameters that include the amplitude and duration of the inflammatory response monitored by the neutrophil infiltration. BCA treatment shortened Ri from ∼23 h observed in vehicle-treated mice to ∼5.5 h, associated with an increase in apoptotic events and efferocytosis, both key steps for the resolution of inflammation. These effects of BCA were prevented by H89, an inhibitor of protein kinase A (PKA) and G15, a selective G protein-coupled receptor 30 (GPR30) antagonist. In line with the in vivo data, BCA also increased the efferocytic ability of murine bone marrow-derived macrophages. Collectively, these data indicate for the first time that BCA resolves neutrophilic inflammation acting in key steps of the resolution of inflammation, requiring activation of GPR30 and via stimulation of cAMP-dependent signaling.

Angiotensin-(1-7) and Alamandine Promote Anti-inflammatory Response in Macrophages In Vitro and In Vivo.

The renin-angiotensin system (RAS) peptides play an important role in inflammation. Resolution of inflammation contributes to restore tissue homeostasis, and it is characterized by neutrophil apoptosis and their subsequent removal by macrophages, which are remarkable plastic cells involved in the pathophysiology of diverse inflammatory diseases. However, the effects of RAS peptides on different macrophage phenotypes are still emerging. Here, we evaluated the effects of angiotensin-(1-7) (Ang-(1-7)) and the most novel RAS peptide, alamandine, on resting (M0), proinflammatory M(LPS+IFN-γ), and anti-inflammatory M(IL-4) macrophage phenotypes in vitro, as well as on specific immune cell populations and macrophage subsets into the pleural cavity of LPS-induced pleurisy in mice. Our results showed that Ang-(1-7) and alamandine, through Mas and MrgD receptors, respectively, do not affect M0 macrophages but reduce the proinflammatory TNF-α, CCL2, and IL-1ß transcript expression levels in LPS+IFN-γ-stimulated macrophages. Therapeutic administration of these peptides in LPS-induced inflammation in mice decreased the number of neutrophils and M1 (F4/80lowGr1+CD11bmed) macrophage frequency without affecting the other investigated macrophage subsets. Our data suggested that both Ang-(1-7) and alamandine, through their respective receptors Mas and MrgD, promote an anti-inflammatory reprogramming of M(LPS+IFN-γ)/M1 macrophages under inflammatory circumstances and potentiate the reprogramming induced by IL-4. In conclusion, our work sheds light on the emerging proresolving properties of Ang-(1-7) and alamandine, opening new avenues for the treatment of inflammatory diseases.

Delay of neuropathic pain sensitization after application of dexamethasone-loaded implant in sciatic nerve-injured rats

Neuroimmune interactions underlying the development of pain sensitization in models of neuropathic pain have been widely studied. In this study, we evaluated the development of allodynia and its reduction associated with peripheral antineuroinflammatory effects induced by a dexamethasone-loaded biodegradable implant. Chronic constriction injury (CCI) of the sciatic nerve was performed in Wistar rats. The electronic von Frey test was applied to assess mechanical allodynia. The dexamethasone-loaded implant was placed perineurally at the moment of CCI or 12 days after surgery. Dorsal root ganglia (DRG; L4-L5) were harvested and nuclear extracts were assayed by Western blot for detection of nuclear factor (NF)-κB p65/RelA translocation. Dexamethasone delivered from the implant delayed the development of allodynia for approximately three weeks in CCI rats when the implantation was performed at day 0, but allodynia was not reversed when the implantation was performed at day 12. NF-κB was activated in CCI rat DRG compared with naïve or sham animals (day 15), and dexamethasone implant inhibited p65/RelA translocation in CCI rats compared with control. This study demonstrated that the dexamethasone-loaded implant suppresses allodynia development and peripheral neuroinflammation. This device can reduce the potential side effects associated with oral anti-inflammatory drugs.

Annexin A1 and the Resolution of Inflammation: Modulation of Neutrophil Recruitment, Apoptosis, and Clearance.

Neutrophils (also named polymorphonuclear leukocytes or PMN) are essential components of the immune system, rapidly recruited to sites of inflammation, providing the first line of defense against invading pathogens. Since neutrophils can also cause tissue damage, their fine-tuned regulation at the inflammatory site is required for proper resolution of inflammation. Annexin A1 (AnxA1), also known as lipocortin-1, is an endogenous glucocorticoid-regulated protein, which is able to counterregulate the inflammatory events restoring homeostasis. AnxA1 and its mimetic peptides inhibit neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis. AnxA1 also promotes monocyte recruitment and clearance of apoptotic leukocytes by macrophages. More recently, some evidence has suggested the ability of AnxA1 to induce macrophage reprogramming toward a resolving phenotype, resulting in reduced production of proinflammatory cytokines and increased release of immunosuppressive and proresolving molecules. The combination of these mechanisms results in an effective resolution of inflammation, pointing to AnxA1 as a promising tool for the development of new therapeutic strategies to treat inflammatory diseases.

Impairment of stress granule assembly via inhibition of the eIF2alpha phosphorylation sensitizes glioma cells to chemotherapeutic agents.

Malignant gliomas are a lethal type of brain tumors that poorly respond to chemotherapeutic drugs. Several therapy resistance mechanisms have been characterized. However, the response to stress through mRNA translational control has not been evaluated for this type of tumor. A potential target would involve the alpha subunit of eukaryotic translation initiation factor (eIF2α) that leads to assembly of stress granules (SG) which are cytoplasmic granules mainly composed by RNA binding proteins and untranslated mRNAs. We assessed whether glioma cells are capable of assembling SG after exposure to different classes of chemotherapeutic agents through evaluation of the effects of interfering in this process by impairing the eIF2α signaling. C6 and U87MG cells were exposed to bortezomib, cisplatin, or etoposide. Forced expression of a dominant negative mutant of eIF2α (eIF2α(DN)) was employed to block this pathway. We observed that exposure to drugs stimulated SG assembly. This was reduced in eIF2α(DN)-transfected cells and this strategy enhanced chemotherapeutically-induced cell death for all drugs. Our data suggest that SG assembly occurs in glioma cells in response to chemotherapeutic drugs in an eIF2α-dependent manner and this response is relevant for drug resistance. Interfering with eIF2α signaling pathway may be a potential strategy for new co-adjuvant therapies to treat gliomas.

PI3Kγ deficiency enhances seizures severity and associated outcomes in a mouse model of convulsions induced by intrahippocampal injection of pilocarpine.

Phosphatidylinositol 3-kinase (PI3K) is an enzyme involved in different pathophysiological processes, including neurological disorders. However, its role in seizures and postictal outcomes is still not fully understood. We investigated the role of PI3Kγ on seizures, production of neurotrophic and inflammatory mediators, expression of a marker for microglia, neuronal death and hippocampal neurogenesis in mice (WT and PI3Kγ(-/-)) subjected to intrahippocampal microinjection of pilocarpine. PI3Kγ(-/-) mice presented a more severe status epilepticus (SE) than WT mice. In hippocampal synaptosomes, genetic or pharmacological blockade of PI3Kγ enhanced the release of glutamate and the cytosolic calcium concentration induced by KCl. There was an enhanced neuronal death and a decrease in the doublecortin positive cells in the dentate gyrus of PI3Kγ(-/-) animals after the induction of SE. Levels of BDNF were significantly increased in the hippocampus of WT and PI3Kγ(-/-) mice, although in the prefrontal cortex, only PI3Kγ(-/-) animals showed significant increase in the levels of this neurotrophic factor. Pilocarpine increased hippocampal microglial immunolabeling in both groups, albeit in the prelimbic, medial and motor regions of the prefrontal cortex this increase was observed only in PI3Kγ(-/-) mice. Regarding the levels of inflammatory mediators, pilocarpine injection increased interleukin (IL) 6 in the hippocampus of WT and PI3Kγ(-/-) animals and in the prefrontal cortex of PI3Kγ(-/-) animals 24h after the stimulus. Levels of TNFα were enhanced in the hippocampus and prefrontal cortex of only PI3Kγ(-/-) mice at this time point. On the other hand, PI3Kγ deletion impaired the increase in IL-10 in the hippocampus induced by pilocarpine. In conclusion, the lack of PI3Kγ revealed a deleterious effect in an animal model of convulsions induced by pilocarpine, suggesting that this enzyme may play a protective role in seizures and pathological outcomes associated with this condition.

Phosphatidylinositol 3-Kinase γ is required for the development of experimental cerebral malaria.

Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3+CD8+ T cells were significantly reduced in the brain of infected PI3Kγ-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.

Switching off key signaling survival molecules to switch on the resolution of inflammation.

Inflammation is a physiological response of the immune system to injury or infection but may become chronic. In general, inflammation is self-limiting and resolves by activating a termination program named resolution of inflammation. It has been argued that unresolved inflammation may be the basis of a variety of chronic inflammatory diseases. Resolution of inflammation is an active process that is fine-tuned by the production of proresolving mediators and the shutdown of intracellular signaling molecules associated with cytokine production and leukocyte survival. Apoptosis of leukocytes (especially granulocytes) is a key element in the resolution of inflammation and several signaling molecules are thought to be involved in this process. Here, we explore key signaling molecules and some mediators that are crucial regulators of leukocyte survival in vivo and that may be targeted for therapeutic purposes in the context of chronic inflammatory diseases.

Altered intracellular signaling cascades in peripheral blood mononuclear cells from BD patients.

Bipolar disorder (BD) is a severe psychiatric disorder of complex physiopathology that has been associated with a pro-inflammatory state. The aim of the present study was to investigate intracellular pathways associated with inflammatory signaling, assessing the phosphorylation levels of transcription factor nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPKs) in peripheral blood mononuclear cells of euthymic BD patients and healthy controls. Fifteen BD euthymic type I patients, and 12 healthy controls matched by age and gender were enrolled in this study. All subjects were assessed by the Mini-International Neuropsychiatry Interview and the patients also by the Young Mania Rating Scale and the Hamilton Depression Rating Scale. Phosphorylation levels of p65 NF-κB subunit, and MAPK ERK1/2, and p38 were assessed by Western blot and flow cytometry. Plasma cytokines (IL-2, IL-4, IL6, IL-10, IFN-γ, TNF-α, and IL-17A) were measured using cytometric bead arrays. Western blot and flow cytometry analyses showed increased phosphorylation levels of p65 NF-κB subunit, and MAPKs ERK1/2, and p38 in BD patients in euthymia in comparison with controls. BD patients presented increased pro-inflammatory cytokines levels in comparison with controls, and TNF-α correlated with the levels of phosphorylated p65 NF-κB. The present study found increased activation of MAPK and NF-κB pathways in BD patients, which is in line with a pro-inflammatory status.

Dengue-3 encephalitis promotes anxiety-like behavior in mice.

Dengue virus is a human pathogen that may cause meningoencephalitis and other neurological syndromes. The current study investigated anxiety-like behavior and expression of proinflammatory cytokines and pro-apoptotic caspase-3 in the hippocampus of C57BL/6 mice infected with non-adapted Dengue virus 3 genotype I (DENV-3) inoculated intracranially with 4×10(3) (plaque-forming unit) PFU. Anxiety-like behavior was assessed in control and DENV-3 infected mice using the elevated plus maze. The open field test was performed to evaluate locomotor activity. Histopathological changes in CA regions of the hippocampus were assessed by haematoxylin and eosin staining. Immunoreactive and protein levels of cleaved caspase-3 were also analyzed in the hippocampus. The mRNA expression of IL-6 and TNF-α in the hippocampus were estimated by quantitative real time (polymerase chain reaction) PCR. All procedures were conducted on day 5 post-infection. We found that DENV-3 infected mice presented higher levels of anxiety in comparison with controls (p≤0.05). No difference in motor activity was found between groups (p=0.77). The infection was followed by a significant increase of TNF-α and IL-6 mRNA expression in the hippocampus (p≤0.05). Histological analysis demonstrated meningoencephalitis with formation of perivascular cuffs, infiltration of immune cells and loss of neurons at CA regions of hippocampus. Numerous caspase-3 positive neurons were visualized at CA areas in DENV-3 infected mice. Marked increase of cleaved caspase-3 levels were observed after infection. This study described anxiety-like behavior, hippocampal inflammation and neuronal apoptosis associated with DENV-3 infection in the central nervous system.