Annexin A1 improves immune responses and control of tissue parasitism during Leishmania amazonensis infection in BALB/c mice.

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.

GILZ Modulates the Recruitment of Monocytes/Macrophages Endowed with a Resolving Phenotype and Favors Resolution of Escherichia coli Infection.

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.

Crosstalk between the plasminogen/plasmin system and inflammation resolution.

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.

A clindamycin acetylated derivative with reduced antibacterial activity inhibits articular hyperalgesia and edema by attenuating neutrophil recruitment, NF-κB activation and tumor necrosis factor-α production.

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

Plasmin and plasminogen prevent sepsis severity by reducing neutrophil extracellular traps and systemic inflammation.

Sepsis is a lethal syndrome characterized by systemic inflammation and abnormal coagulation. Despite therapeutic advances, sepsis mortality remains substantially high. Herein, we investigated the role of the plasminogen/plasmin (Plg/Pla) system during sepsis. Plasma levels of Plg were significantly lower in mice subjected to severe compared with nonsevere sepsis, whereas systemic levels of IL-6, a marker of sepsis severity, were higher in severe sepsis. Plg levels correlated negatively with IL-6 in both septic mice and patients, whereas plasminogen activator inhibitor-1 levels correlated positively with IL-6. Plg deficiency render mice susceptible to nonsevere sepsis induced by cecal ligation and puncture (CLP), resulting in greater numbers of neutrophils and M1 macrophages, liver fibrin(ogen) deposition, lower efferocytosis, and increased IL-6 and neutrophil extracellular trap (NET) release associated with organ damage. Conversely, inflammatory features, fibrin(ogen), and organ damage were substantially reduced, and efferocytosis was increased by exogenous Pla given during CLP- and LPS-induced endotoxemia. Plg or Pla protected mice from sepsis-induced lethality and enhanced the protective effect of antibiotics. Mechanistically, Plg/Pla-afforded protection was associated with regulation of NET release, requiring Pla-protease activity and lysine binding sites. Plg/Pla are important host-protective players during sepsis, controlling local and systemic inflammation and collateral organ damage.

Ouratea spectabilis and its Biflavanone Ouratein D Exert Potent Anti-inflammatory Activity in MSU Crystal-induced Gout in Mice.

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

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.

Relevance of angiotensin-(1-7) and its receptor Mas in pneumonia caused by influenza virus and post-influenza pneumococcal infection.

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.

Blame the signaling: Role of cAMP for the resolution of inflammation.

A complex intracellular signaling governs different cellular responses in inflammation. Extracellular stimuli are sensed, amplified, and transduced through a dynamic cellular network of messengers converting the first signal into a proper response: production of specific mediators, cell activation, survival, or death. Several overlapping pathways are coordinated to ensure specific and timely induction of inflammation to neutralize potential harms to the tissue. Ideally, the inflammatory response must be controlled and self-limited. Resolution of inflammation is an active process that culminates with termination of inflammation and restoration of tissue homeostasis. Comparably to the onset of inflammation, resolution responses are triggered by coordinated intracellular signaling pathways that transduce the message to the nucleus. However, the key messengers and pathways involved in signaling transduction for resolution are still poorly understood in comparison to the inflammatory network. cAMP has long been recognized as an inducer of anti-inflammatory responses and cAMP-dependent pathways have been extensively exploited pharmacologically to treat inflammatory diseases. Recently, cAMP has been pointed out as coordinator of key steps of resolution of inflammation. Here, we summarize the evidence for the role of cAMP at inducing important features of resolution of inflammation.

Harnessing inflammation resolving-based therapeutic agents to treat pulmonary viral infections: What can the future offer to COVID-19?

Inflammation is generally accepted as a component of the host defence system and a protective response in the context of infectious diseases. However, altered inflammatory responses can contribute to disease in infected individuals. Many endogenous mediators that drive the resolution of inflammation are now known. Overall, mediators of resolution tend to decrease inflammatory responses and provide normal or greater ability of the host to deal with infection. In the lung, it seems that pro-resolution molecules, or strategies that promote their increase, tend to suppress inflammation and lung injury and facilitate control of bacterial or viral burden. Here, we argue that the demonstrated anti-inflammatory, pro-resolving, anti-thrombogenic and anti-microbial effects of such endogenous mediators of resolution may be useful in the treatment of the late stages of the disease in patients with COVID-19.

Glucocorticoid-induced leucine zipper modulates macrophage polarization and apoptotic cell clearance.

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.

The role of annexin A1 in the modulation of the NLRP3 inflammasome.

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.

Cyclic AMP Regulates Key Features of Macrophages via PKA: Recruitment, Reprogramming and Efferocytosis.

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-γ/LPS-induced macrophage polarization to M1-like as shown by increased Arg-1 associated to lower levels of M1 cytokines (TNF-α/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.

Plasminogen and the Plasminogen Receptor, Plg-RKT, Regulate Macrophage Phenotypic, and Functional Changes.

Inflammation resolution is an active process that functions to restore tissue homeostasis. Clearance of apoptotic leukocytes by efferocytosis at inflammatory sites plays an important role in inflammation resolution and induces remarkable macrophage phenotypic and functional changes. Here, we investigated the effects of deletion of either plasminogen (Plg) or the Plg receptor, Plg-RKT, on the resolution of inflammation. In a murine model of pleurisy, the numbers of total mononuclear cells recruited to the pleural cavity were significantly decreased in both Plg-/- and Plg-RKT-/- mice, a response associated with decreased levels of the chemokine CCL2 in pleural exudates. Increased percentages of M1-like macrophages were determined in pleural lavages of Plg-/- and Plg-RKT-/- mice without significant changes in M2-like macrophage percentages. In vitro, Plg and plasmin (Pla) increased CD206/Arginase-1 expression and the levels of IL-10/TGF-ß (M2 markers) while decreasing IFN/LPS-induced M1 markers in murine bone-marrow-derived macrophages (BMDMs) and human macrophages. Furthermore, IL4-induced M2-like polarization was defective in BMDMs from both Plg-/- and Plg-RKT-/- mice. Mechanistically, Plg and Pla induced transient STAT3 phosphorylation, which was decreased in Plg-/- and Plg-RKT-/- BMDMs after IL-4 or IL-10 stimulation. The extents of expression of CD206 and Annexin A1 (important for clearance of apoptotic cells) were reduced in Plg-/- and Plg-RKT-/- macrophage populations, which exhibited decreased phagocytosis of apoptotic neutrophils (efferocytosis) in vivo and in vitro. Taken together, these results suggest that Plg and its receptor, Plg-RKT, regulate macrophage polarization and efferocytosis, as key contributors to the resolution of inflammation.

Leptin, hsCRP, TNF-α and IL-6 levels from normal aging to dementia: Relationship with cognitive and functional status.

Cognitive impairment, including mild cognitive impairment (MCI) and dementia, compromises the patients' cognitive abilities and, to different extents, to carry out daily activities, accompanied by personality and behavioral changes. Studies suggest that leptin, an adipokine, has a neuroprotective role against Alzheimer's disease (AD) and that cytokines are associated with inflammatory processes and dementia. This study aimed to evaluate serum leptin, hsCRP, IL-6 and TNF-α levels in a cognitive continuum group from normal to demential status, and to assess whether they correlates to Mini-Mental State Examination (MMSE) and Functional Assessment Staging (FAST) scores. Forty-three participants were included, of whom 12 with probable AD, 18 with MCI and 13 with no objective cognitive decline. Serum leptin and hsCRP levels were evaluated by immunoturbidimetric method, and IL-6 and TNF-α by ELISA. Higher TNF-α levels were found in individuals with FAST stages 1/2 and normal scores evaluated by MMSE. hsCRP levels were inversely correlated with FAST stages. No association with function or global cognition was observed for leptin and IL-6 levels. However, women presented higher leptin serum levels than men while lower leptin and IL-6 levels were observed in individuals aged ≥59 years. Our results suggest that TNF-α is associated with cognitive and functional decline and that inflammation could be a substrate of cognitive impairment at early clinical stages of dementia.

Phosphatidyl Inositol 3 Kinase-Gamma Balances Antiviral and Inflammatory Responses During Influenza A H1N1 Infection: From Murine Model to Genetic Association in Patients.

Influenza A virus (IAV) infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ), mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO) mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single-nucleotide polymorphisms (SNPs), rs1129293, rs17847825, and rs2230460. We observed that SNPs rs17847825 and rs2230460 (A and T alleles, respectively) were significantly associated with protection from severe disease using the recessive model in patients infected with influenza A(H1N1)pdm09. Altogether, our results suggest that PI3Kγ is crucial in balancing antiviral and inflammatory responses to IAV infection.

The resolution of acute inflammation induced by cyclic AMP is dependent on annexin A1.

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.

Annexin A1 Is Involved in the Resolution of Inflammatory Responses during Leishmania braziliensis Infection.

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.

Plasmin and plasminogen induce macrophage reprogramming and regulate key steps of inflammation resolution via annexin A1.

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.

Annexin A1 promotes timely resolution of inflammation in murine gout.

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.