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Phosphoinositide-3-kinases (PI3Ks) are enzymes involved in signalling and modification of the function of all mammalian cells. These enzymes phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositol, resulting in lipid products that act as second messengers responsible for coordinating many cellular functions, including activation, chemotaxis, proliferation and survival. The identification of the functions that are mediated by a specific PI3K isoform is complex and depends on the specific cell type and inflammatory context. In this chapter we will focus on the role of PI3K isoforms in the context of innate immunity, focusing on the mechanisms by which PI3K signalling regulates phagocytosis, the activation of immunoglobulin, chemokine and cytokines receptors, production of ROS and cell migration, and how PI3K signalling plays a central role in host defence against infections and tissue injury.

Gout is an inflammatory disease triggered by deposition of monosodium urate (MSU) crystals in the joints, resulting in high neutrophil influx and pain. Here, we studied the role of the inhibitory receptor CD300a in the resolution process in a murine model of gout. We found increased CD300a expression on neutrophils emigrated to the joint. When compared to WT mice, CD300a(−/−) mice had persistent neutrophil influx till 24 hr after MSU injection. This was associated with increased concentration of IL‐1β and greater tissue damage in the joints of CD300a(−/−) mice. There was an increase in the percentage of apoptotic neutrophils in the synovial lavage of WT mice, as compared to CD300a(−/−) mice. This difference was reflected in the decline of efferocytic events in the synovial cavity of CD300a(−/−) mice 24 hr after MSU injection. A CD300a agonistic antibody was shown, for the first time, to increase apoptosis of human neutrophils, and this was associated with cleavage of caspase‐8. In conclusion, our results reveal an important role of CD300a in the control of leucocyte infiltration, IL‐1β production and caspase‐8 cleavage in neutrophils, contributing to the resolution of inflammation triggered by MSU injection.

Background There is mounting evidence that SARS-CoV-2 targets tissues beyond the respiratory tract. Long-term sequelae after COVID-19 are frequent and of major concern. Prolonged virus detection in the gut has been particularly intriguing. Of note, SARS-CoV-2 infection also disturbs the gut microbiota composition, a finding linked with disease severity in patients with COVID-19. Here, we aimed to characterize the functional role of the gut microbiota in the long-term consequences of COVID-19. To this end, we characterized the gut microbiota from COVID-19 human subjects and followed the effects of human fecal transfer to germ-free mice. Results The gut microbiota of post-COVID subjects (up to 4 months from the initial positive test) revealed a remarkable predominance of Enterobacteriaceae strains with multidrug-resistance phenotype compared to healthy controls. After fecal transfer to germ-free mice, animals receiving samples from post-COVID subjects displayed higher lung inflammation and increased susceptibility to pulmonary infection caused by an antimicrobial resistant Klebsiella pneumoniae strain. These mice also showed poorer cognitive performance associated with increased expression of TNF-α, reduced levels of brain-derived neurotrophic factor-BDNF and postsynaptic density protein-PSD-95 in the brain, as well as alterations of several biochemical pathways. These alterations were observed in the absence of SARS-CoV-2, suggesting that alterations in the gut microbiota caused them. Consistent with this hypothesis, brain dysfunctions induced in a mouse model of coronavirus infection were partially prevented by modulation of the microbiota via treatment with the commensal probiotic bacteria Bifidobacterium longum 51A. Conclusions Our results show prolonged impact of SARS-CoV-2 infection in the gut microbiota that persists even after the individuals have cleared the virus. Increased Enterobacteriaceae with antimicrobial resistance phenotype were of particular concern. Moreover, microbiota transfer from post-COVID subjects induced loss of brain cognitive functions and impaired lung defense in mice. Altogether, our work emphasizes the importance of microbiota as a target for therapies to help treat post-COVID sequelae.

The respiratory tract is constantly exposed to environmental aggressions including allergens, infections, and pollution. The immune system is pivotal to fight potential invaders and prevent the establishment of infections in the respiratory tract; however, excessive, misplaced or altered immune responses contribute to increase tissue damage, impair lung function, and exacerbate respiratory diseases. Indeed, asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis are respiratory diseases that have uncontrolled inflammation as a unifying severity factor. In addition, the acute inflammatory responses in pneumonia must be controlled to avoid progression to acute respiratory distress syndrome, which can be lethal. In this regard, immunomodulatory therapies are promising pharmacological strategies to treat important respiratory pathologies. Understanding the pathogenesis of respiratory disorders have paved the way for the development of cytokine-directed therapies, allosteric or non-allosteric inhibitors of inflammatory receptors and enzymes, agonists of resolution of inflammation and other anti-inflammatory compounds. A multitude of studies have evaluated the safety, pharmacokinetics, and pharmacodynamics of several molecules that target inflammation in the respiratory tract, yet only few have been translated to clinical use. This article will summarize important aspects of asthma, COPD, cystic fibrosis, and pneumonia focusing on the role of immune responses in disease development and novel immunomodulatory therapies.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of morbidity and death worldwide. Inhalation of cigarette smoke (CS) is the major cause in developed countries. Current therapies have limited efficacy in controlling disease or halting its progression. Aberrant expression of microRNAs (miRNAs) is associated with lung disease, including COPD. We performed miRNA microarray analyses of the lungs of mice with CS-induced experimental COPD. miR-21 was the second highest up-regulated miRNA, particularly in airway epithelium and lung macrophages. Its expression in human lung tissue correlated with reduced lung function in COPD. Prophylactic and therapeutic treatment with a specific miR-21 inhibitor (Ant-21) inhibited CS-induced lung miR-21 expression in mice; suppressed airway macrophages, neutrophils, and lymphocytes; and improved lung function, as evidenced by decreased lung hysteresis, transpulmonary resistance, and tissue damping in mouse models of COPD. In silico analyses identified a potential miR-21/special AT-rich sequence–binding protein 1 (SATB1)/S100 calcium binding protein A9 (S100A9)/nuclear factor κB (NF-κB) axis, which was further investigated. CS exposure reduced lung SATB1 in a mouse model of COPD, whereas Ant-21 treatment restored SATB1 and reduced S100A9 expression and NF-κB activity. The beneficial effects of Ant-21 in mice were reversed by treatment with SATB1-targeting small interfering RNA. We have identified a pathogenic role for a miR-21/SATB1/S100A9/NF-κB axis in COPD and defined miR-21 as a therapeutic target for this disease.

Macrophages are a heterogeneous population of myeloid cells with phenotype and function modulated according to the microenvironment in which they are found. The lung resident macrophages known as Alveolar Macrophages (AM) and Interstitial Macrophages (IM) are localized in two different compartments. During lung homeostasis, macrophages can remove inhaled particulates, cellular debris and contribute to some metabolic processes. Macrophages may assume a pro-inflammatory phenotype after being classically activated (M1) or anti-inflammatory when being alternatively activated (M2). M1 and M2 have different transcription profiles and act by eliminating bacteria, viruses and fungi from the host or repairing the damage triggered by inflammation, respectively. Nevertheless, macrophages also may contribute to lung damage during persistent inflammation or continuous exposure to antigens. In this review, we discuss the origin and function of pulmonary macrophages in the context of homeostasis, infectious and non-infectious lung diseases.

The gastrointestinal tract encounters a wide variety of microorganisms, including beneficial symbionts, pathobionts, and pathogens. Recent evidence has shown that the gut microbiota, directly or indirectly through its components, such as metabolites, actively participates in the host inflammatory response by cytokine-microbiota or microbiota-cytokine modulation interactions, both in the gut and systemically. Therefore, further elucidation of host cytokine molecular pathways and microbiota components will provide a novel and promising therapeutic approach to control or prevent inflammatory disease and to maintain host homeostasis. The purpose of this review is to summarize well-established scientific findings and provide an updated overview regarding the direct and indirect mechanisms by which the gut microbiota can influence the inflammatory response by modulating the host's cytokine pathways that are mostly involved, but not exclusively so, with gut homeostasis. In addition, we will highlight recent results from our group, which suggest that the microbiota promotes cytokine release from inflammatory cells though activation of microbial metabolite sensor receptors that are more highly expressed on inflammatory and intestinal epithelial cells.

Idiopathic pulmonary fibrosis is characterized by aberrant fibroblast activation and excessive collagen deposition that may eventually lead to organ dysfunction. Lung fibrosis is frequently observed in cancer patients undergoing bleomycin (BLM) treatment. Therefore, BLM instillation in mice is the most frequent model used to investigate pulmonary fibrosis. Angiotensin 1–7 [Ang-(1-7)] is a heptapeptide with anti-inflammatory and proresolving activity. Here, we studied the effects of preventive and therapeutic oral administration of Ang-(1-7) in a model of BLM-induced lung fibrosis in mice. Male C57Bl/6j mice were instilled with BLM and followed for weight loss and survival or euthanized to examine pulmonary inflammation, fibrosis, and lung function. For preventive treatment, mice were treated with Ang-(1-7) 1 h before instillation and then twice daily. We observed that preventive treatment with Ang-(1-7) decreased weight loss, inflammation and collagen deposition, increased survival, and ameliorated lung function. Therapeutic treatment with Ang-(1-7), starting 3 days after BLM instillation resulted in decreased inflammation, decreased collagen deposition, and ameliorated lung function, although the effects were of lower magnitude than the preventive treatment. Therapeutic treatment with Ang-(1-7) starting 7 or 14 days after BLM instillation failed to alter any of the changes observed. Therefore, although oral preventive treatment with Ang-(1-7) is effective to decrease pulmonary inflammation, fibrosis, and functional changes induced by BLM, therapeutic effects are much less significant, arguing against its use in patients with chronic fibrosis. It remains to be determined whether other proresolving molecules will have better therapeutic effects in the context of chronic pulmonary fibrosis.

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.

Brucella abortus is a Gram-negative bacterium responsible for a worldwide zoonotic infection—Brucellosis, which has been associated with high morbidity rate in humans and severe economic losses in infected livestock. The natural route of infection is through oral and nasal mucosa but the invasion process through host gut mucosa is yet to be understood. Studies have examined the role of NLRP6 (NOD-like receptor family pyrin domain-containing-6 protein) in gut homeostasis and defense against pathogens. Here, we investigated the impact of gut microbiota and NLRP6 in a murine model of Ba oral infection. Nlrp6-/- and wild-type (WT) mice were infected by oral gavage with Ba and tissues samples were collected at different time points. Our results suggest that Ba oral infection leads to significant alterations in gut microbiota. Moreover, Nlrp6-/- mice were more resistant to infection, with decreased CFU in the liver and reduction in gut permeability when compared to the control group. Fecal microbiota transplantation from WT and Nlrp6-/- into germ-free mice reflected the gut permeability phenotype from the donors. Additionally, depletion of gut microbiota by broad-spectrum-antibiotic treatment prevented Ba replication in WT while favoring bacterial growth in Nlrp6-/-. Finally, we observed higher eosinophils in the gut and leukocytes in the blood of infected Nlrp6-/- compared to WT-infected mice, which might be associated to the Nlrp6-/- resistance phenotype. Altogether, these results indicated that gut microbiota composition is the major factor involved in the initial stages of pathogen host replication and partially also by the resistance phenotype observed in Nlrp6 -/- mice regulating host inflammation against Ba infection., Author summary Brucella abortus (Ba) is an intracellular bacterium that causes zoonotic and clinical problems worldwide. Although the common route of infection is through oral and nasal, the mechanisms toward the gastrointestinal mucosa response is still unexplored. It is well known that microbiota promotes and maintains host intestinal homeostasis during bacterial infections. However, mechanisms by which the gut microbiota affects the Ba infection have not yet been demonstrated. Here, we provide significant insights into the relationship between gut microbiota and B. abortus oral infection and demonstrate the gut microbiota contribution to the gut permeability and dissemination of Ba. Furthermore, we investigated the participation of the gut microbiota from Nlrp6 deficient mice, on the gut permeability and Ba infection. Substantial experiments performed, mostly in vivo, showed that gut microbiota alterations promote gut barrier disruption, as indicated by increased gut permeability after Ba oral infection. Thus, our work highlights the role of gut mucosal environment through gut microbiota and Nlrp6 molecule involved in host innate immune responses to Ba infection.

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.

Mucositis is an inflammation of the gastrointestinal mucosa resulting from high doses of radio/chemotherapy treatment and may lead to interruption of antineoplasic therapy. Soluble fibres, like pectin, increase SCFA production, which play a role in gut homoeostasis and inflammation suppression. Due to the properties of pectin, the aim of the present study was to evaluate the effect of a high-fibre (HF) diet on chemotherapy-induced mucositis in a murine model. C57/BL6 mice received control (AIN93M), HF, low/zero fibre (LF) diets for 10 d prior to mucositis challenging with irinotecan (75 mg/kg), or they were treated with acetate added to drinking water 5 d prior to and during the mucositis induction. Mice that received the HF diet showed decreased immune cells influx and improved histopathological parameters in the intestine, compared with mice that received the normal diet. Furthermore, the HF diet decreased intestinal permeability induced in the mucositis model when compared with the control group. This effect was not observed for acetate alone, which did not improve gut permeability. For instance, mice that received the LF diet had worsened gut permeability, compared with mice that received the normal diet and mucositis. The effects of the HF and LF diets were shown to modulate the intestinal microbiota, in which the LF diet increased the levels of Enterobacteriaceae, a group associated with gut inflammation, whereas the HF diet decreased this group and increased Lactobacillus and Bifidobacterium (SCFA producers) levels. In conclusion, the results demonstrated the importance of dietary fibre intake in the modulation of gut microbiota composition and homoeostasis maintenance during mucositis in this model.

Asthma is a heterogeneous disease with complex multifactorial causes. It is possible to subclassify asthma into different phenotypes that have distinct immunological features. Eosinophilic asthma is a well-known phenotype of severe asthma; however, a large body of clinical and experimental evidence strongly associates persistent airway inflammation, including the accumulation of neutrophils in the bronchial mucosa, and resistance to corticosteroid therapy and non-Type-2 immune responses with severe asthma. Importantly, mainstay therapies are often ineffective in severe asthma and effective alternatives are urgently needed.Here, we discussed recently developed mouse models of severe asthma that recapitulates key features of the disease in humans. We also provide findings from clinically relevant experimental models that have identified potential therapeutic targets for severe asthma. The most relevant publications on the topic of interest were selected from PubMed.Increasing the understanding of disease-causing mechanisms in severe asthma may lead to the identification of novel therapeutic targets and the development of more effective therapies. Intense research interest into investigating the pathophysiological mechanisms of severe asthma has driven the development and interrogation of a myriad of mouse models that aim to replicate hallmark features of severe asthma in humans.

Annexins are well‐known Ca(2+ )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)‐1β 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‐1β release in vivo and in vitro. These effects were due to reduction of transcriptional levels of IL‐1β, 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.

Staphylococcus aureus is the main cause of septic arthritis in humans, a disease associated with high morbidity and mortality. Inflammation triggered in response to infection is fundamental to control bacterial growth but may cause permanent tissue damage. Here, we evaluated the role of Lipoxin A4 (LXA4 ) in S aureus-induced arthritis in mice. Septic arthritis was induced by S aureus injection into tibiofemoral joints. At different time points, we evaluated cell recruitment and bacterial load in the joint, the production of pro-inflammatory molecules, and LXA4 in inflamed tissue and analyzed joint damage and dysfunction. LXA4 was investigated using genetically modified mice or by pharmacological blockade of its synthesis and receptor. CD11c+ cells were evaluated in lymph nodes by confocal microscopy and flow cytometry and dendritic cell chemotaxis using the Boyden chamber. Absence or pharmacological blockade of 5-lipoxygenase (5-LO) reduced joint inflammation and dysfunction and was associated with better control of infection at 4 to 7 days after the infection. There was an increase in LXA4 in joints of S aureus-infected mice and administration of LXA4 reversed the phenotype in 5-LO-/- mice. Blockade or absence of the LXA4 receptor FPR2 has a phenotype similar to 5-LO-/- mice. Mechanistically, LXA4 appeared to control migration and function of dendritic cells, cells shown to be crucial for adequate protective responses in the model. Thus, after the first days of infection when symptoms become evident therapies that inhibit LXA4 synthesis or action could be useful for treatment of S aureus-induced arthritis.

Rheumatoid arthritis(RA) is a debilitating chronic inflammatory disease. Suppressors of Cytokine Signaling(SOCS) proteins regulate homeostasis and pathogenesis in several diseases. The intersection between RA pathophysiology and SOCS2 is unclear. Herein, we investigated the roles of SOCS2 during the development of an experimental antigen-induced arthritis(AIA). In wild type mice, joint SOCS2 expression was reduced during AIA development. At the peak of inflammation, SOCS2-/- mice presented with reduced numbers of infiltrated cells in their joints. At the late phase of AIA, however, exhibited increased adhesion/infiltration of neutrophils, macrophages, CD4+-T cells, CD4+CD8+-T cells, and CD4-CD8--T cells associated with elevated IL-17 and IFN-γ levels, joint damage, proteoglycan loss, and nociception. SOCS2 deficiency resulted in lower numbers of apoptotic neutrophils and reduced efferocytosis. The present study demonstrated the vital role of SOCS2 during the development and resolution of an experimental RA model. Hence, this protein may be a novel therapeutic target for this disorder.

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-1β, and CXCL1 production in periarticular joint. Posttreatment with Ac2-26 decreased neutrophil accumulation, IL-1β, 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.

Mice are widely used to assess the pathogenesis of diseases. An experimental model of gout consists of the injection of uric acid crystals into joints of mice, which reproduce inflammation and functional changes of the human disease. Uric acid crystals activate synoviocytes culminating in the release of IL-1β and neutrophil recruitment, key inflammatory elements in gouty arthritis. Since MIF plays an important role in orchestrating gout inflammation, we detail valuable procedures to investigate uric acid crystal-induced joint inflammation in mice and give options for further understanding the functions of MIF in gouty arthritis in vivo and in vitro.

Mice are widely used to assess the pathogenesis of diseases. An experimental model of gout consists of the injection of uric acid crystals into joints of mice, which reproduce inflammation and functional changes of the human disease. Uric acid crystals activate synoviocytes culminating in the release of IL-1β and neutrophil recruitment, key inflammatory elements in gouty arthritis. Since MIF plays an important role in orchestrating gout inflammation, we detail valuable procedures to investigate uric acid crystal-induced joint inflammation in mice and give options for further understanding the functions of MIF in gouty arthritis in vivo and in vitro.

Uncontrolled inflammation leads to tissue damage and it is central for the development of chronic inflammatory diseases and autoimmunity. An acute inflammatory response is finely regulated by the action of anti-inflammatory and pro-resolutive mediators, culminating in the resolution of inflammation and restoration of homeostasis. There are few studies investigating intracellular signaling pathways associated with the resolution of inflammation. Here, we investigate the role of Rho-associated kinase (ROCK), a serine/threonine kinase, in a model of self-resolving neutrophilic inflammatory. We show that ROCK activity, evaluated by P-MYPT-1 kinetics, was higher during the peak of lipopolysaccharide-induced neutrophil influx in the pleural cavity of mice. ROCK inhibition by treatment with Y-27632 decreased the accumulation of neutrophils in the pleural cavity and was associated with an increase in apoptotic events and efferocytosis, as evaluated by an in vivo assay. In a model of gout, treatment with Y-27632 reduced neutrophil accumulation, IL-1&beta, levels and hypernociception in the joint. These were associated with reduced MYPT and I&kappa, B&alpha, phosphorylation levels and increased apoptosis. Finally, inhibition of ROCK activity also induced apoptosis in human neutrophils and destabilized cytoskeleton, extending the observed effects to human cells. Taken together, these data show that inhibition of the ROCK pathway might represent a potential therapeutic target for neutrophilic inflammatory diseases.

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-1β 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.

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-1β 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.

Ang-(1-7) binds to the G-protein-coupled MAS receptor and exerts many beneficial actions in the context of acute inflammation. Ang-(1-7) reduces production and expression of many cytokines and adhesion molecules and decreases leukocyte movement in vitro and in vivo. In animal models of inflammation, administration of Ang-(1-7) or its analogues is, in general, associated with inhibition of leukocyte influx and inflammatory tissue damage. In addition, Ang-(1-7) reduces fibrogenesis in the context of chronic inflammation. Ang-(1-7) also induces the resolution of inflammation by inducing apoptosis of neutrophils and their clearance by macrophages. Altogether, the available literature suggests that drugs that mimic the action of Ang-(1-7) on the MAS receptor may represent a novel pharmacological strategy to control and treat inflammatory diseases.

Staphylococcus aureus is the main cause of septic arthritis in humans, a disease associated with high morbidity and mortality. Inflammation triggered in response to infection is fundamental to control bacterial growth but may cause permanent tissue damage. Here, we evaluated the role of Lipoxin A

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-1β 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.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais INCT – Instituto nacional de ciência e tecnologia (Antigo Instituto do Milênio) Gout is a disease characterized by the deposition of monosodium urate (MSU) crystals in the joints. Continuous gout episodes may lead to unresolved inflammatory responses and tissue damage. We investigated the effects of a high-fiber diet and acetate, a short-chain fatty acid (SCFA) resulting from the metabolism of fiber by gut microbiota, on the inflammatory response in an experimental model of gout in mice. Injection of MSU crystals into the knee joint of mice induced neutrophil influx and inflammatory hypernociception. The onset of inflammatory response induced by MSU crystals was not altered in animals given a high-fiber diet, but the high-fiber diet induced faster resolution of the inflammatory response. Similar results were obtained in animals given the SCFA acetate. Acetate was effective, even when given after injection of MSU crystals at the peak of the inflammatory response and induced caspase-dependent apoptosis of neutrophils that accounted for the resolution of inflammation. Resolution of neutrophilic inflammation was associated with decreased NF-κB activity and enhanced production of anti-inflammatory mediators, including IL-10, TGF-β, and annexin A1. Acetate treatment or intake of a high-fiber diet enhanced efferocytosis, an effect also observed in vitro with neutrophils treated with acetate. In conclusion, a high-fiber diet or one of its metabolic products, acetate, controls the inflammatory response to MSU crystals by favoring the resolution of the inflammatory response. Our studies suggest that what we eat plays a determinant role in our capacity to fine tune the inflammatory response.

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.

Gout is an acute inflammatory disease characterised by the presence of uric acid crystals in the joint. This event promotes neutrophil infiltration and activation that leads to tissue damage. We investigated here whether the oral administration of the probiotic strain Bifidobacterium longum 51A(BL) could ameliorate monosodium urate crystal (MSU)-induced inflammation in a murine model of gout. Mice received oral administration of BL or saline daily for 7 days and then were injected with MSU in the knee cavity. Treatment with BL significantly alleviated the inflammatory parameters, as seen by reduced hypernociception, reduced neutrophil accumulation in the joint and myeloperoxidase activity in periarticular tissue. There was inhibition of the production of CXCL1 and interleukin(IL)-1β in joints. Levels of the anti-inflammatory cytokine IL-10 were significantly higher in the knee tissue of mice treated with than control mice injected with MSU. In conclusion, oral BL treatment reduced the inflammatory response in an experimental murine model of gout, suggesting it may be useful as an adjuvant treatment in patients with gout.

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.

Macrophages are central to inflammation resolution, an active process aimed at restoring tissue homeostasis following an inflammatory response. Here, the effects of db-cAMP on macrophage phenotype and function were investigated. Injection of db-cAMP into the pleural cavity of mice induced monocytes recruitment in a manner dependent on PKA and CCR2/CCL2 pathways. Furthermore, db-cAMP promoted reprogramming of bone-marrow-derived macrophages to a M2 phenotype as seen by increased Arg-1/CD206/Ym-1 expression and IL-10 levels (M2 markers). Db-cAMP also showed a synergistic effect with IL-4 in inducing STAT-3 phosphorylation and Arg-1 expression. Importantly, db-cAMP prevented IFN-&gamma, /LPS-induced macrophage polarization to M1-like as shown by increased Arg-1 associated to lower levels of M1 cytokines (TNF-&alpha, /IL-6) and p-STAT1. In vivo, db-cAMP reduced the number of M1 macrophages induced by LPS injection without changes in M2 and Mres numbers. Moreover, db-cAMP enhanced efferocytosis of apoptotic neutrophils in a PKA-dependent manner and increased the expression of Annexin A1 and CD36, two molecules associated with efferocytosis. Finally, inhibition of endogenous PKA during LPS-induced pleurisy impaired the physiological resolution of inflammation. Taken together, the results suggest that cAMP is involved in the major functions of macrophages, such as nonphlogistic recruitment, reprogramming and efferocytosis, all key processes for inflammation resolution.

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.

Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes that are involved in many aspects of immune cell function. PI3Kγ and PI3Kδ are the major isoforms expressed in leukocytes. The role of PI3K isoforms in the resolution of inflammation is still poorly understood. Here, we investigated the contribution of PI3Kγ and PI3Kδ to the resolution of inflammation in a model of gout in mice. Methods and Results: Experiments were performed in wild-type male C57/Bl6 mice. Selective inhibitors of PI3K-γ (AS605240) or PI3Kδ (GSK045) were injected in the joint 12 h after injection of MSU crystals, hence at the peak of inflammation. Inhibition of either PI3K isoform decreased number of neutrophils that migrated in response to the injection of MSU crystals. This was associated with reduction of myeloperoxidase activity and IL-1β levels in periarticular tissues and reduction of histological score. Joint dysfunction, as seen by reduced mechanical hypernociception, was improved by treatment with either inhibitor. The decrease in neutrophil numbers was associated with enhanced apoptosis and efferocytosis of these cells. There was shortening of resolution intervals, suggesting inhibition of either isoform induced the resolution of neutrophilic inflammation. Blockade of PI3Kγ or PI3Kδ reduced Nuclear Factor kappa B (NF-κB) activation. A pan-PI3K inhibitor (CL27c) reduced inflammation induced by MSU crystals by a magnitude that was similar to that attained by the PI3Kγ or PI3Kδ selective inhibitors alone. Conclusion: Taken together, these results suggest that neutrophils can use PI3Kγ or PI3Kδ to remain in the cavity and blockade of either isoenzyme is sufficient to induce their apoptosis and resolve inflammation in a murine model of gout.

Pneumonia is one of the leading causes of death and mortality worldwide. The inflammatory responses that follow respiratory infections are protective leading to pathogen clearance but can also be deleterious if unregulated. The microbiota is known to be an important protective barrier against infections, mediating both direct inhibitory effects against the potential pathogen and also regulating the immune responses contributing to a proper clearance of the pathogen and return to homeostasis. GPR43 is one receptor for acetate, a microbiota metabolite shown to induce and to regulate important immune functions. Here, we addressed the role of GPR43 signaling during pulmonary bacterial infections. We have shown for the first time that the absence of GPR43 leads to increased susceptibility to Klebsiella pneumoniae infection, which was associated to both uncontrolled proliferation of bacteria and to increased inflammatory response. Mechanistically, we showed that GPR43 expression especially in neutrophils and alveolar macrophages is important for bacterial phagocytosis and killing. In addition, treatment with the GPR43 ligand, acetate, is protective during bacterial lung infection. This was associated to reduction in the number of bacteria in the airways and to the control of the inflammatory responses. Altogether, GPR43 plays an important role in the “gut–lung axis” as a sensor of the host gut microbiota activity through acetate binding promoting a proper immune response in the lungs.

Inflammation is a physiologic response against noxious stimuli and microbial invaders. The basic elements of inflammation include host cells, blood vessels, proteins and lipid mediators, which work together to eliminate the inflammatory stimulus as well as initiate the resolution and repair. Mediators of inflammation are regulatory molecules that control the generation, maintenance and resolution of this response, which is triggered after recognition of infection or injure. The initial recognition of the inflammatory stimuli leads to the production of pro-inflammatory mediators. These mediators are derived from immune cells (e.g. vasoactive amines, lipid mediators, platelet-activating factor, reactive oxygen species, nitric oxide, cytokines and chemokines) or are acute phase proteins produced by the liver that circulate in the plasma (e.g. the complement, coagulation and kallikrein-kinin systems). Together, the mediators of inflammation orchestrate all the inflammatory events such as blood vessel dilatation, vascular permeability, leukocyte migration to the affected tissue and pain.