GPCRs overexpression and impaired fMLP-induced functions in neutrophils from chronic kidney disease patients

INTRODUCTION: G-protein coupled receptors (GPCRs) expressed on neutrophils regulate their mobilization from the bone marrow into the blood, their half-live in the circulation, and their pro- and anti-inflammatory activities during inflammation. Chronic kidney disease (CKD) is associated with systemic inflammatory responses, and neutrophilia is a hallmark of CKD onset and progression. Nonetheless, the role of neutrophils in CKD is currently unclear. METHODS: Blood and renal tissue were collected from non-dialysis CKD (grade 3 - 5) patients to evaluate GPCR neutrophil expressions and functions in CKD development. RESULTS: CKD patients presented a higher blood neutrophil-to-lymphocyte ratio (NLR), which was inversely correlated with the glomerular filtration rate (eGFR). A higher frequency of neutrophils expressing the senescent GPCR receptor (CXCR4) and activation markers (CD18+CD11b+CD62L+) was detected in CKD patients. Moreover, CKD neutrophils expressed higher amounts of GPCR formyl peptide receptors (FPR) 1 and 2, known as neutrophil pro- and anti-inflammatory receptors, respectively. Cytoskeletal organization, migration, and production of reactive oxygen species (ROS) by CKD neutrophils were impaired in response to the FPR1 agonist (fMLP), despite the higher expression of FPR1. In addition, CKD neutrophils presented enhanced intracellular, but reduced membrane expression of the protein Annexin A1 (AnxA1), and an impaired ability to secrete it into the extracellular compartment. Secreted and phosphorylated AnxA1 is a recognized ligand of FPR2, pivotal in anti-inflammatory and efferocytosis effects. CKD renal tissue presented a low number of neutrophils, which were AnxA1+. CONCLUSION: Together, these data highlight that CKD neutrophils overexpress GPCRs, which may contribute to an unbalanced aging process in the circulation, migration into inflamed tissues, and efferocytosis.

Lack of Annexin A1 Exacerbates Inflammatory Response in Acute Endometritis Model.

Annexin A1 (AnxA1) is a glucocorticoid-inducible protein and an important endogenous modulator of inflammation. However, its effect in the endometrial microenvironment is poorly explained. This study aimed to evaluate the role of endogenous AnxA1 in an endometritis mouse model induced by lipopolysaccharide (LPS). Female C57BL/6 wild-type (WT) and AnxA1-/- mice were divided into two groups: SHAM and LPS. To induce endometritis, mice received a vaginal infusion of 50 µL of LPS (1 mg/mL) dissolved in phosphate-buffered saline. After 24 h, the mice were euthanized, and blood and uteri samples were collected. The endometrium inflammatory scores were significantly increased in the LPS-treated group. AnxA1-/- mice from the LPS group demonstrated a significant increase in the number of degranulated mast cell levels compared to AnxA1-/- SHAM mice. The Western blotting analysis revealed that a lack of AnxA1 promoted the upregulation of NLRP3 and pro-IL-1ß in the acute endometritis animal model compared to WT LPS animals. LPS-induced endometritis increased the number of blood peripheral leukocytes in both WT and AnxA1-/- mice compared with SHAM group mice (p < 0.001). AnxA1-/- mice also showed increased plasma levels of IL-1ß (p < 0.01), IL-6, IL-10, IL-17, and TNF-α (p < 0.05) following LPS-induced endometritis. In conclusion, a lack of endogenous AnxA1 exacerbated the inflammatory response in an endometritis model via NLRP3 dysregulation, increased uterine mast cell activation, and plasma pro-inflammatory cytokine release.

Annexin A12-26 hydrogel improves healing properties in an experimental skin lesion after induction of type 1 diabetes.

Diabetes mellitus (DM) is characterized by metabolic alterations that involve defects in the secretion and/or action of insulin, being responsible for several complications, such as impaired healing. Studies from our research group have shown that annexin A1 protein (AnxA1) is involved in the regulation of inflammation and cell proliferation. In light of these findings, we have developed a new technology and evaluated its effect on a wound healing in vivo model using type 1 diabetes (T1DM)-induced mice. We formulated a hydrogel containing AnxA12-26 using defined parameters such as organoleptic characteristics, pH, UV-vis spectroscopy and cytotoxicity assay. UV-vis spectroscopy confirmed the presence of the associated AnxA12-26 peptide in the three-dimensional hydrogel matrix, while the in vitro cytotoxicity assay showed excellent biocompatibility. Mice showed increased blood glucose levels, confirming the efficacy of streptozotocin (STZ) to induce T1DM. Treatment with AnxA12-26 hydrogel showed to improve diabetic wound healing, defined as complete re-epithelialization and tissue remodeling, with reduction of inflammatory infiltrate in diabetic animals. We envisage that the AnxA12-26 hydrogel, with its innovative composition and formulation be efficient on improving diabetic healing and contributing on the expansion of the therapeutic arsenal to treat diabetic wounds, at a viable cost.

Annexin A1, A2, A5, and A6 involvement in human pathologies.

The human genome codes for 12 annexins with highly homologous membrane-binding cores and unique amino termini, which endow each protein with its specific biological properties. Not unique to vertebrate biology, multiple annexin orthologs are present in almost all eukaryotes. Their ability to combine either dynamically or constitutively with membrane lipid bilayers is hypothetically the key property that has led to their retention and multiple adaptation in eukaryotic molecular cell biology. Annexin genes are differentially expressed in many cell types but their disparate functions are still being discovered after more than 40 years of international research. A picture is emerging from gene knock down and knock out studies of individual annexins that these are important supporters rather than critical players in organism development and normal cell and tissue function. However, they appear to be highly significant "early responders" toward challenges arising from cell and tissue abiotic or biotic stress. In humans, recent focus has been on involvement of the annexin family for its involvement in diverse pathologies, especially cancer. From what has become an exceedingly broad field of investigation, we have selected four annexins in particular: AnxA1, 2, 5, and 6. Present both within and external to cells, these annexins are currently under intensive investigation in translational research as biomarkers of cellular dysfunction and as potential therapeutic targets for inflammatory conditions, neoplasia, and tissue repair. Annexin expression and release in response to biotic stress appears to be a balancing act. Under- or over-expression in different circumstances appears to damage rather than restore a healthy homeostasis. This review reflects briefly on what is already known of the structures and molecular cell biology of these selected annexins and considers their actual and potential roles in human health and disease.

Bisphenol A disruption promotes mammary tumor microenvironment via phenotypic cell polarization and inflammatory response.

Inflammation in the established tumor microenvironment (TME) is often associated with a poor prognosis of breast cancer. Bisphenol A (BPA) is an endocrine-disrupting chemical that acts as inflammatory promoter and tumoral facilitator in mammary tissue. Previous studies demonstrated the onset of mammary carcinogenesis at aging when BPA exposure occurred in windows of development/susceptibility. We aim to investigate the inflammatory repercussions of BPA in TME in mammary gland (MG) during neoplastic development in aging. Female Mongolian gerbils were exposed to low (50 µg/kg) or high BPA (5000 µg/kg) doses during pregnancy and lactation. They were euthanized at 18 months of age (aging) and the MG were collected for inflammatory markers and histopathological analysis. Contrarily to control MG, BPA induced carcinogenic development mediated by COX-2 and p-STAT3 expression. BPA was also able to promote macrophage and mast cell (MC) polarization in tumoral phenotype, evidenced by pathways for recruitment and activation of these inflammatory cells and tissue invasiveness triggered by tumor necrosis factor-alpha and transforming growth factor-beta 1 (TGF-ß1). Increase of tumor-associated macrophages, M1 (CD68 + iNOS+) and M2 (CD163+) expressing pro-tumoral mediators and metalloproteases was observed; this aspect greatly contributed to stromal remodeling and invasion of neoplastic cells. In addition, the MC population drastically increased in BPA-exposed MG. Tryptase-positive MCs increased in disrupted MG and expressed TGF-ß1, contributing to EMT process during carcinogenesis mediated by BPA. BPA exposure interfered in inflammatory response by releasing and enhancing the expression of mediators that contribute to tumor growth and recruitment of inflammatory cells that promote a malignant profile.

Galectin-3 is a key hepatoprotective molecule against the deleterious effect of cisplatin.

AIMS: Evaluate the role of galectin-3 in the liver using an acute model of cisplatin-induced toxicity. MATERIAL AND METHODS: Modified citrus pectin (MCP) treatment was used to inhibit galectin-3. Rats were distributed into four groups: SHAM, CIS, MCP and MCP + CIS. On days 1-7, animals were treated by oral gavage with 100 mg/kg/day of MCP (MCP and MCP + CIS groups). On days 8, 9 and 10, animals received intraperitoneal injection of 10 mg/kg/day of cisplatin (CIS and MCP + CIS groups) or saline (SHAM and MCP groups). KEY FINDINGS: Cisplatin administration caused a marked increase in hepatic leukocyte influx and liver degeneration, and promoted reactive oxygen species production and STAT3 activation in hepatocytes. Plasma levels of cytokines (IL-6, IL-10), and hepatic toxicity biomarkers (hepatic arginase 1, α-glutathione S-transferase, sorbitol dehydrogenase) were also elevated. Decreased galectin-3 levels in the livers of animals in the MCP + CIS group were also associated with increased hepatic levels of malondialdehyde and mitochondrial respiratory complex I. Animals in the MCP + CIS group also exhibited increased plasma levels of IL-1ß, TNF-α, and aspartate transaminase 1. Furthermore, MCP therapy efficiently antagonized hepatic galectin-9 in liver, but not galectin-1, the latter of which was increased. SIGNIFICANCE: Reduction of the endogenous levels of galectin-3 in hepatocytes favors the process of cell death and increases oxidative stress in the acute model of cisplatin-induced toxicity.

Effects of galectin-3 protein on UVA-induced damage in retinal pigment epithelial cells.

Several inflammatory molecules have been suggested as biomarkers of age-related macular degeneration (AMD). Galectin-3 (Gal-3), which has been shown to have a protective role in corneal injury by promoting epithelial cells adhesion and migration to the extracellular matrix, is also highly expressed in the retinal pigment epithelium (RPE) of patients with AMD. This study evaluated the role of Gal-3 in an in vitro model of UVA-induced RPE damage, as a proof-of-concept. ARPE-19 cells (human RPE cell line), were incubated with Gal-3 at 0.5-2.5 µg/mL concentrations prior to UVA irradiation for 15, 30, and 45 min, which resulted in accumulated doses of 2.5, 5, and 7.5 J/cm2, respectively. After 24 h incubation, MTT and LDH assays, immunofluorescence, and ELISA were performed. UVA irradiation for 15, 30, and 45 min proved to reduce viability in 83%, 46%, and 11%, respectively. Based on the latter results, we chose the intermediate dose (5-J/cm2) for further analysis. Pretreatment with Gal-3 at concentrations > 1.5 µg/mL showed to increase the viability of UVA-irradiated cells (~ 75%) compared to untreated cells (64%). Increased levels of cleaved caspase 3, a marker of cell death, were detected in the ARPE cells after UVA irradiation with or without addition of exogenous Gal-3. The inhibitory effect of Gal-3 on UVA-induced cell damage was characterized by decreased ROS levels and increased p38 activation, as detected by fluorescence analysis. In conclusion, our study suggests a photoprotective effect of Gal-3 on RPE by reducing oxidative stress and increasing p38 activation.

Annexins as potential targets in ocular diseases.

Annexins (AnxAs) are Ca2+/phospholipid-binding proteins extensively studied and generally involved in several diseases. Although evidence exists regarding the distribuition of AnxAs in the visual system, their exact roles and the exact cell types of the eye where these proteins are expressed are not well-understood. AnxAs have pro-resolving roles in infectious, autoimmune, degenerative, fibrotic and angiogenic conditions, making them an important target in ocular tissue homeostasis. This review summarizes the current knowledge on the distribution and function of AnxA1-8 isoforms under normal and pathological conditions in the visual system, as well as perspectives for ophthalmologic treatments, including the potential use of the AnxA1 recombinant and/or its mimetic peptide Ac2-26.

The role of annexin A1-derived peptide Ac2-26 on liver and kidney injuries induced by cisplatin in rats.

AIMS: In this study we evaluated the effect of pharmacological treatment with AnxA1-derived peptide Ac2-26 in an experimental model of toxicity induced by cisplatin. MAIN METHODS: Male rats were divided into Sham (control), Cisplatin (received intraperitoneal injections of 10 mg/kg/day of cisplatin for 3 days) and Ac2-26 (received intraperitoneal injections of 1 mg/kg/day of peptide, 15 min before cisplatin) groups. KEY FINDINGS: After 6 h of the last dose of cisplatin, an acute inflammatory response was observed characterized by a marked increase in the number of neutrophils and GM-CSF, IL-ß, IL-6, IL-10 and TNF-α plasma levels. These findings were associated with increased AnxA1 protein levels in liver and kidneys, as well as positive AnxA1/Fpr2 circulating leukocytes. Treatment with Ac2-26 produced higher levels of GM-CSF, corroborating the high numbers of neutrophils, and the anti-inflammatory cytokine IL-4. Ac2-26 preserved the morphology of liver structures and increased Fpr1 expression, preventing the damage caused by cisplatin. In the kidneys, Ac2-26 caused downregulation of renal Fpr1 and Fpr2 levels and abrogated the increased levels of the CLU and KIM-1 biomarkers of kidney damage induced by cisplatin. However, no effect of peptide treatment was detected in cisplatin-induced kidney morphology injury. SIGNIFICANCE: Despite activation of the anti-inflammatory AnxA1/Fpr axis during cisplatin administration, treatment with Ac2-26 did not efficiently prevent its deleterious effects on the liver and kidneys.

Pharmacological treatment with annexin A1-derived peptide protects against cisplatin-induced hearing loss.

Cisplatin is an antineoplastic agent widely used, and no effective treatments capable of preventing cisplatin-induced ototoxicity and neurotoxicity in humans have yet been identified. This study evaluated the effect of the anti-inflammatory annexin A1 (AnxA1)-derived peptide Ac2-26 in a cisplatin-induced ototoxicity model. Wistar rats received intraperitoneal injections of cisplatin (10 mg/kg/day) for 3 days to induce hearing loss, and Ac2-26 (1 mg/kg) was administered 15 min before cisplatin administration. Control animals received an equal volume of saline. Hearing thresholds were measured by distortion product otoacoustic emissions (DPOAE) before and after treatments. Pharmacological treatment with Ac2-26 protected against cisplatin-induced hearing loss, as evidenced by DPOAE results showing similar signal-noise ratios between the control and Ac2-26-treated groups. These otoprotective effects of Ac2-26 were associated with an increased number of ganglion neurons compared with the untreated cisplatin group. Additionally, Ac2-26 treatment produced reduced immunoreactivity on cleaved caspase 3 and phosphorylated ERK levels in the ganglion neurons, compared to the untreated group, supporting the neuroprotective effects of the Ac2-26. Our results suggest that Ac2-26 has a substantial otoprotective effect in this cisplatin-induced ototoxicity model mediated by neuroprotection and the regulation of the ERK pathway.

Effects of Formyl Peptide Receptor Agonists Ac9-12 and WKYMV in In Vivo and In Vitro Acute Inflammatory Experimental Models.

Formyl peptide receptors (Fprs) are a G-protein-coupled receptor family mainly expressed on leukocytes. The activation of Fpr1 and Fpr2 triggers a cascade of signaling events, leading to leukocyte migration, cytokine release, and increased phagocytosis. In this study, we evaluate the effects of the Fpr1 and Fpr2 agonists Ac9-12 and WKYMV, respectively, in carrageenan-induced acute peritonitis and LPS-stimulated macrophages. Peritonitis was induced in male C57BL/6 mice through the intraperitoneal injection of 1 mL of 3% carrageenan solution or saline (control). Pre-treatments with Ac9-12 and WKYMV reduced leukocyte influx to the peritoneal cavity, particularly neutrophils and monocytes, and the release of IL-1ß. The addition of the Fpr2 antagonist WRW4 reversed only the anti-inflammatory actions of WKYMV. In vitro, the administration of Boc2 and WRW4 reversed the effects of Ac9-12 and WKYMV, respectively, in the production of IL-6 by LPS-stimulated macrophages. These biological effects of peptides were differently regulated by ERK and p38 signaling pathways. Lipidomic analysis evidenced that Ac9-12 and WKYMV altered the intracellular lipid profile of LPS-stimulated macrophages, revealing an increased concentration of several glycerophospholipids, suggesting regulation of inflammatory pathways triggered by LPS. Overall, our data indicate the therapeutic potential of Ac9-12 and WKYMV via Fpr1 or Fpr2-activation in the inflammatory response and macrophage activation.

Expression Pattern and Immunoregulatory Roles of Galectin-1 and Galectin-3 in Atopic Dermatitis and Psoriasis.

The pathogenesis of atopic dermatitis (AD) and psoriasis (Ps) overlaps, particularly the activation of the immune response and tissue damage. Here, we evaluated galectin (Gal)-1 and Gal-3 levels, which are beta-galactoside-binding proteins with immunomodulatory functions and examined their effects on human keratinocytes stimulated with either interleukin (IL)-4 or IL-17A. Skin biopsies from AD, Ps, and control patients were evaluated using histological and immunohistochemical analyses. Six studies containing publicly available transcriptome data were individually analyzed using the GEO2R tool to detect Gal-1 and Gal-3 mRNA levels. In vitro, IL-4- or IL-17A-stimulated keratinocytes were treated with or without Gal-1 or Gal-3 to evaluate cytokine release and migration. Our findings showed different patterns of expression for Gal-1 and Gal-3 in AD and Ps skins. Densitometric analysis in skin samples showed a marked increase in the protein Gal-1 levels in Ps epidermis and in both AD and Ps dermis compared to controls. Protein and mRNA Gal-3 levels were downregulated in AD and Ps lesional skin compared with the control samples. In vitro, both galectins addition abrogated the release of IL-8 and RANTES in IL-17-stimulated keratinocytes after 24 h, whereas IL-6 release was downregulated by Gal-3 and Gal-1 in IL-4- and IL-17-stimulated cells, respectively. Administration of both galectins also increased the rate of keratinocyte migration under IL-4 or IL-17 stimulation conditions compared with untreated cells. Altogether, the immunoregulatory and migration effects of Gal-1 and Gal-3 on keratinocytes under inflammatory microenvironment make them interesting targets for future therapies in cutaneous diseases.

Bellis perennis extract mitigates UVA-induced keratinocyte damage: Photoprotective and immunomodulatory effects.

A need exists for further research elucidating the benefits of environmentally safe photoprotective agents against ultraviolet (UV) exposure, and plant extracts represent a human-friendly alternative formulation. This study was designed to evaluate the potential use of Bellis perennis extract (BPE), from the Asteraceae family, known as the common daisy or the English daisy, in cosmeceuticals as a photoprotective factor, using an in vitro model of UVA-induced keratinocyte damage. Human skin keratinocytes (HaCaT cell line) were incubated with BPE at 0.01, 0.1, or 1% in Dulbecco's Modified Eagle Medium (DMEM), and after 15 min they were submitted to UVA radiation at 5, 10, and 15 J/cm2 doses, respectively. For comparative purposes, Polypodium leucotomos extract (PLE), known as the fern, was used as a positive control in assessing the photoprotective effect. After 24 h of UVA exposure, cell viability (MTT and LDH assays), levels of cleaved caspase-3, cyclooxygenase-2, IL-6, reactive oxygen species (ROS) and antioxidant enzyme (catalase, SOD, and glutathione peroxidase) activity were determined. UVA radiation at 5, 10, and 15 J/cm2 doses reduced cell viability to 63%, 43%, and 23%, respectively; we selected 10 J/cm2 for our purposes. After 24 h of UVA exposure, treatment with 1% BPE and 1% PLE significantly recovered cell viability (p < 0.05). Furthermore, treatment was associated with lower cleaved caspase-3 and ROS levels, higher catalase activity, and lower IL-6 levels in the treated UVA keratinocytes compared with the untreated UVA group (p < 0.01). Our results demonstrate photoprotective and immunomodulatory effects of BPE in skin keratinocytes and support its use as a bioactive agent in cosmetic formulations to prevent skin damage caused by exposure to the UV light.

The Role of Galectin-9 as Mediator of Atopic Dermatitis: Effect on Keratinocytes.

Galectin-9 (Gal-9) is a beta-galactoside-binding protein with a variety of biological functions related to immune response. However, in allergic diseases, its mechanism of action is not fully understood. This study evaluates the expression pattern of Gal-9 in patients with atopic dermatitis (AD), in ovalbumin (OVA)-induced experimental atopic dermatitis (AD) in mice, as well as its effect on human keratinocytes. The skin of OVA-immunized BALB/c mice was challenged with drops containing OVA on days 11, 14-18, and 21-24. HaCaT cells were cultured in the following experimental conditions: control (growth medium only) or stimulated with TNF-α/IFN-γ, or IL-4, or IL-17 with or without Gal-9 treatment. AD was characterized by increased levels of Gal-9 in mouse and human skin, especially in the epidermis, and with a marked influx of Gal-9 positive eosinophils and mast cells compared to the control group. Gal-9 showed an immunomodulatory effect on keratinocytes by decreasing the release of IL-6 by IL-4-stimulated keratinocytes or increasing the IL-6 and RANTES levels by IL-17- or TNF-α/IFN-γ-stimulated cells, respectively. Under IL-17, Gal-9 treatment also altered the proliferation rate of cells. Overall, increased levels of Gal-9 in AD skin contribute to the control of inflammatory response and the proliferative process of keratinocytes, suggesting this lectin as a relevant therapeutic target.

Role of Annexin A1 in NLRP3 Inflammasome Activation in Murine Neutrophils.

This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1ß production by WT neutrophils after nigericin and ATP stimulation. However, IL-1ß release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1ß release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1ß production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.

Annexin A1-derived peptide Ac2-26 in a pilocarpine-induced status epilepticus model: anti-inflammatory and neuroprotective effects.

BACKGROUND: The inflammatory process has been described as a crucial mechanism in the pathophysiology of temporal lobe epilepsy. The anti-inflammatory protein annexin A1 (ANXA1) represents an interesting target in the regulation of neuroinflammation through the inhibition of leukocyte transmigration and the release of proinflammatory mediators. In this study, the role of the ANXA1-derived peptide Ac2-26 in an experimental model of status epilepticus (SE) was evaluated. METHODS: Male Wistar rats were divided into Naive, Sham, SE and SE+Ac2-26 groups, and SE was induced by intrahippocampal injection of pilocarpine. In Sham animals, saline was applied into the hippocampus, and Naive rats were only handled. Three doses of Ac2-26 (1 mg/kg) were administered intraperitoneally (i.p.) after 2, 8 and 14 h of SE induction. Finally, 24 h after the experiment-onset, rats were euthanized for analyses of neuronal lesion and inflammation. RESULTS: Pilocarpine induced generalised SE in all animals, causing neuronal damage, and systemic treatment with Ac2-26 decreased neuronal degeneration and albumin levels in the hippocampus. Also, both SE groups showed an intense influx of microglia, which was corroborated by high levels of ionised calcium binding adaptor molecule 1(Iba-1) and monocyte chemoattractant protein-1 (MCP-1) in the hippocampus. Ac2-26 reduced the astrocyte marker (glial fibrillary acidic protein; GFAP) levels, as well as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and growth-regulated alpha protein (GRO/KC). These effects of the peptide were associated with the modulation of the levels of formyl peptide receptor 2, a G-protein-coupled receptor that binds to Ac2-26, and the phosphorylated extracellular signal-regulated kinase (ERK) in the hippocampal neurons. CONCLUSIONS: The data suggest a neuroprotective effect of Ac2-26 in the epileptogenic processes through downregulation of inflammatory mediators and neuronal loss.

Pharmacological treatment with galectin-1 protects against renal ischaemia-reperfusion injury.

Galectin-1 protein (GAL-1) has important anti-inflammatory properties, but related pharmacologic approaches to effectively treat or prevent renal ischaemia and reperfusion injury are highly limited. Here, we investigated the effect of GAL-1 in a renal ischaemia-reperfusion injury rat model and an in vitro hypoxia-reoxygenation model with a proximal renal tubular epithelial cell line. In vivo, pretreatment with GAL-1 attenuated the renal parameters changed by ischaemia-reperfusion/hypoxia-reoxygenation, with recovery of renal function, protecting against influx of leukocytes, cell death and oxidative stress. Ischaemia-reperfusion/hypoxia-reoxygenation was also associated with increased renal endogenous expression of GAL-1 and intercellular adhesion molecule 1 (ICAM-1) plus augmented levels of proinflammatory cytokines IL-1ß, TNF-α and MCP-1 and decreased anti-inflammatory IL-10 in urine, all of which were abrogated by GAL-1 treatment. In vitro studies demonstrated renal tubular epithelial cells as an important source of GAL-1 during hypoxia-reoxygenation and confirmed the protective effects of exogenous GAL-1 through downregulation of proinflammatory cytokine release by proximal renal tubular epithelial cells. Collectively, our findings confirm the important anti-inflammatory role of GAL-1 in kidney ischaemia and reperfusion injury and indicate its promising use as a therapeutic approach.

Beneficial effect of annexin A1 in a model of experimental allergic conjunctivitis.

Annexin A1 (ANXA1), a 37 kDa glucocorticoid-regulated protein, is a potent anti-inflammatory mediator effective in terminating acute inflammatory response, and its role in allergic settings has been poorly studied. The aim of this investigation was to evaluate the mechanism of action of ANXA1 in intraocular inflammation using a classical model of ovalbumin (OVA)-induced allergic conjunctivitis (AC). OVA-immunised Balb/c mice, wild-type (WT) and ANXA1-deficient (AnxA1(-/-)), were challenged with eye drops containing OVA on days 14-16 with a subset of WT animals pretreated intraperitoneally with the peptide Ac2-26 (N-terminal region of ANXA1) or dexamethasone (DEX). After 24 h of the last ocular challenge, WT mice treated with Ac2-26 and DEX had significantly reduced clinical signs of conjunctivitis (chemosis, conjunctival hyperaemia, lid oedema and tearing), plasma IgE levels, leukocyte (eosinophil and neutrophil) influx and mast cell degranulation in the conjunctiva compared to WT controls. These anti-inflammatory effects of DEX were associated with high endogenous levels of ANXA1 in the ocular tissues as detected by immunohistochemistry. Additionally, Ac2-26 administration was effective to reduce IL-2, IL-4, IL-10, IL-13, eotaxin and RANTES in the eye and lymph nodes compared to untreated WT animals. The lack of ANXA1 produced an exacerbated allergic response as detected by the density of the inflammatory cell influx to the conjunctiva and the cytokine/chemokine release. These different effects observed for Ac2-26 were correlated with diminished level of activated ERK at 24 h in the ocular tissues compared to untreated OVA group. Our findings demonstrate the protective effect of ANXA1 during the inflammatory allergic response suggesting this protein as a potential target for new ocular inflammation therapies.

The intricate role of mast cell proteases and the annexin A1-FPR1 system in abdominal wall endometriosis.

Endometriosis is a continuous and progressive disease with a poorly understood aetiology, pathophysiology and natural history. This study evaluated the histological differences between eutopic and ectopic endometria (abdominal wall endometriosis) and the expression of mast cell proteases (tryptase and chymase), annexin A1 (ANXA1) and formyl peptide receptor 1 (FPR1). Ectopic endometrium from 18 women with abdominal wall endometriosis and eutopic endometrium from 10 women without endometriosis were obtained. The endometrial samples were analysed by histopathology, immunohistochemistry and ultrastructural immunogold labeling to determine mast cell heterogeneity (tryptase and chymase positive cells) and the expression levels of ANXA1 and FPR1. Histopathological analysis of the endometriotic lesions showed a glandular pattern of mixed differentiation and an undifferentiated morphology with a significant influx of inflammatory cells and a change in mast cell heterogeneity, as evidenced by a significant increase in the number of chymase-positive cells and endogenous chymase expression. The undifferentiated glandular pattern of endometriotic lesions was positively associated with a marked increase and co-localization of ANXA1 and FPR1 in the epithelial cells. In conclusion, the co-upregulated expression of mast cell chymase and ANXA1-FPR1 system in ectopic endometrium suggests their involvement in the development of endometriotic lesions.

Anti-inflammatory mechanisms of the annexin A1 protein and its mimetic peptide Ac2-26 in models of ocular inflammation in vivo and in vitro.

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1(-/-) mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach.