Maresin 1, a proresolving lipid mediator derived from omega-3 polyunsaturated fatty acids, exerts protective actions in murine models of colitis.

It has been previously reported that dietary fish oils, which are rich in the polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid, can exert beneficial effects in inflammatory bowel disease. In this study, we investigated the effects of docosahexaenoic acid-derived lipid mediator maresin 1 (MaR1) in dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice. Systemic treatment with MaR1 significantly attenuated both DSS- and 2,4,6-trinitrobenzene sulfonic acid-induced colonic inflammation by improving the disease activity index and reducing body weight loss and colonic tissue damage. MaR1 treatment also induced a significant decrease in levels of inflammatory mediators, such as IL-1ß, TNF-α, IL-6, and IFN-γ, in the acute protocol, as well as IL-1ß and IL-6, but not TNF-α and INF-γ, in the chronic DSS colitis protocol. Additionally, MaR1 decreased ICAM-1 mRNA expression in both the acute and chronic protocols of DSS-induced colitis. Furthermore, the beneficial effects of MaR1 seem to be associated with inhibition of the NF-κB pathway. Moreover, incubation of LPS-stimulated bone marrow-derived macrophage cultures with MaR1 reduced neutrophil migration and reactive oxygen species production, besides decreasing IL-1ß, TNF-α, IL-6, and INF-γ production. Interestingly, macrophages incubated only with MaR1 showed a significant upregulation of mannose receptor C, type 1 mRNA expression, an M2 macrophage phenotype marker. These results indicate that MaR1 consistently protects mice against different models of experimental colitis, possibly by inhibiting the NF-κB pathway and consequently multiple inflammatory mediators, as well as by enhancing the macrophage M2 phenotype.

The role of kinin B1 and B2 receptors in the persistent pain induced by experimental autoimmune encephalomyelitis (EAE) in mice: evidence for the involvement of astrocytes.

Multiple sclerosis (MS) is a progressive, demyelinating inflammatory disease of the human central nervous system (CNS). While the primary symptoms of MS affect motor function, it is now recognized that chronic pain is a relevant symptom that affects both animals and MS patients. There is evidence that glial cells, such as astrocytes, play an important role in the development and maintenance of chronic pain. Kinins, notably bradykinin (BK) acting through B1 (B1R) and B2 (B2R) receptors, play a central role in pain and inflammatory processes. However, it remains unclear whether kinin receptors are involved in neuropathic pain in MS. Here we investigated by genetic and pharmacological approaches the role of kinin receptors in neuropathic pain behaviors induced in the experimental autoimmune encephalomyelitis (EAE) mouse model. Our results showed that gene deletion or antagonism of kinin receptors, especially B1R, significantly inhibited both tactile and thermal hypersensitivity in EAE animals. By contrast, animals with EAE and treated with a B1R selective agonist displayed a significant increase in tactile hypersensitivity. We also observed a marked increase in B1R mRNA and protein level in the mouse spinal cord 14days after EAE immunization. Blockade of B1R significantly suppressed the levels of mRNAs for IL-17, IFN-γ, IL-6, CXCL-1/KC, COX-2 and NOS2, as well as glial activation in the spinal cord. Of note, the selective B1 antagonist DALBK consistently prevented IFN-induced up-regulation of TNF-α and IL-6 release in astrocyte culture. Finally, both B1R and B2R antagonists significantly inhibited COX-2 and NOS2 expression in primary astrocyte culture. The B1R was co-localized with immunomarker of astrocytes in the spinal cord of EAE-treated animals. The above data constitute convincing experimental evidence indicating that both kinin receptors, especially the B1 subtype, exert a critical role in the establishment of persistent hypersensitivity observed in the EAE model, an action that seems to involve a central inflammatory process, possibly acting on astrocytes. Thus, B1 selective antagonists or drugs that reduce kinin release may have the potential to treat neuropathic pain in patients suffering from MS.

ABCC transporter inhibition reduces zymosan-induced peritonitis.

Inflammatory mediators are released from injured tissues being responsible for the first steps of inflammatory processes. Multidrug efflux transporters, members of the ATP-binding cassette (ABC) family, are ubiquitously expressed. ABCC molecules transport several endogenous substances, including leukotriene C4 (LTC4) and PGE2, which are involved in zymosan-induced inflammation. The present study investigated the role played by ABCC transporters on zymosan-induced peritonitis in mice. Most of the resident peritoneal cells were macrophages, based on their morphology and membrane-activated complex 3 expression. RT-PCR demonstrated that these cells expressed ABCC, and ABCC activity was analyzed in vivo via the s.c. injection of ABCC inhibitors [probenecid (PROB) 200 mg/kg or MK571 20 mg/kg], followed by an i.v. injection of carboxyfluorescein diacetate (CFDA), an ABCC fluorescent substrate. Both inhibitors increased CFDA accumulation, suggesting ABCC impairment. Moreover, ABCC reversors decreased zymosan-induced plasma exudation by 86.6 +/- 7.4 and 97.6 +/- 2.3%, a feature related to a diminished secretion of LTC(4) (65.1+/-11 and 47.8+/-9.9%) and PGE(2) (under basal levels). Cell migration was inhibited similarly. Furthermore, PROB and MK571 inhibited IL-1ss by 83.4 +/- 13 and 71.2 +/- 13.4% and TNF-alpha content by 47 +/- 4.5 and 28.9 +/- 0.8%, respectively. NO metabolites and reactive oxygen species production were also reduced. The present results suggest that ABCC molecules have a relevant role in the acute inflammatory response produced by zymosan in mice.

Multidrug resistance related protein (ABCC1) and its role on nitrite production by the murine macrophage cell line RAW 264.7.

Multidrug resistance related protein 1 (MRP1/ABCC1) is an ABC transporter protein related to the extrusion of reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH-conjugates, as well as leukotriene C(4) and cyclopentane prostaglandins. Inhibition of ABCC1 activity impairs lymphocyte activation. The present work studied ABCC1 expression and activity on a murine macrophage cell line, RAW 267.4 and the effects of ABCC1 classical inhibitors, as well as GSH metabolism modulators, on LPS induced activation. Approximately, 75% of resting cells were positive for ABCC1 and the classical ABCC1 reversors (indomethacin, 0.1-2mM; probenecid, 0.1-10mM and MK571, 0.01-1mM) were able to enhance intracellular CFDA accumulation in a concentration-dependent manner, suggesting ABCC1 inhibition. After LPS (100ng/ml) activation 50% of the population was positive for ABCC1, and this protein was still active. In LPS-activated cells, ABCC1 activity was also impaired by BSO (1mM), an inhibitor of GSH synthesis. Conversely, GSH (5mM) reversed the BSO effect. ABCC1 inhibition by indomethacin, probenecid or MK571 decreased LPS induced nitrite production in a concentration-dependent manner, the same result was observed with BSO and again GSH reversed its effect. The ABCC1 reversors were also able to inhibit iNOS expression. In conclusion, LPS modulated the expression and activity of ABCC1 transporters in RAW macrophages and inhibitors of these transporters were capable of inhibiting nitrite production suggesting a role for ABCC1 transporters in the inflammatory process.

In vivo and in vitro modulation of MDR molecules in murine thymocytes.

P-glycoprotein (Pgp/ABCB1) and multidrug resistance related protein 1 (MRP1/ABCC1) were first described in multidrug resistant tumor cells. It is presently known that both proteins are also expressed in a variety of normal cells, including lymphocytes. ABCB1 activity has already been detected in subpopulations of murine thymocytes, but there was little information on the expression or activity of ABCC1 in these cells. The present work studied in mice the expression of both proteins by RT-PCR and immunofluorescence. It was possible to identify the presence of ABCB1 and to detect the expression of ABCC1 in these cells. The functional activities of these proteins were also studied in vivo and in vitro measuring the extrusion of fluorescent dyes in association with MDR modulators. Cyclosporine A, verapamil and trifluoperazine inhibited the activity of thymic ABCB1. Indomethacin, probenecid and MK571 were effective in inhibiting ABCC1 activity by thymic cells. ABCB1 was only active in a small percentage of thymocytes being present in the immature double negative (not CD4 nor CD8) subpopulation and the mature single positive (CD4 or CD8) subpopulations. The functional activity of ABCC1, on the other hand, was more homogeneously distributed being found in all thymocyte subpopulations. Possible physiological roles for these transporters on thymocytes are discussed.

The role of kinin receptors in preventing neuroinflammation and its clinical severity during experimental autoimmune encephalomyelitis in mice.

BACKGROUND: Multiple sclerosis (MS) is a demyelinating and neuroinflammatory disease of the human central nervous system (CNS). The expression of kinins is increased in MS patients, but the underlying mechanisms by which the kinin receptor regulates MS development have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Experimental autoimmune encephalomyelitis (EAE) was induced in female C57BL/6 mice by immunization with MOG(35-55) peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Here, we report that blockade of the B(1)R in the induction phase of EAE markedly suppressed its progression by interfering with the onset of the immune response. Furthermore, B(1)R antagonist suppressed the production/expression of antigen-specific T(H)1 and T(H)17 cytokines and transcription factors, both in the periphery and in the CNS. In the chronic phase of EAE, the blockade of B(1)R consistently impaired the clinical progression of EAE. Conversely, administration of the B(1)R agonist in the acute phase of EAE suppressed disease progression and inhibited the increase in permeability of the blood-brain barrier (BBB) and any further CNS inflammation. Of note, blockade of the B(2)R only showed a moderate impact on all of the studied parameters of EAE progression. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest that kinin receptors, mainly the B(1)R subtype, play a dual role in EAE progression depending on the phase of treatment through the lymphocytes and glial cell-dependent pathways.

Preventive and therapeutic anti-inflammatory properties of the sesquiterpene alpha-humulene in experimental airways allergic inflammation.

BACKGROUND AND PURPOSE: alpha-Humulene and trans-caryophyllene are plant sesquiterpenes with pronounced anti-inflammatory properties. Here, we evaluated the effects of these compounds in an experimental model of airways allergic inflammation. EXPERIMENTAL APPROACH: Female BALB/c mice, sensitized to and challenged with ovalbumin received daily alpha-humulene or trans-caryophyllene (50 mg.kg(-1), orally) or alpha-humulene (1 mg.mL(-1), by aerosol) as either a preventive (for 22 days) or therapeutic (from the 18th to the 22nd day) treatment. Dexamethasone or budesonide was used as a positive control drug. Inflammation was determined on day 22 post-immunization by leukocyte recruitment, interleukin-5 (IL-5), CCL11, interferon-gamma (IFN-gamma) and leukotriene (LT)B(4) levels in bronchoalveolar lavage fluid (BALF). In addition, transcription factors [nuclear factor kappaB (NF-kappaB), activator protein 1 (AP-1)] and P-selectin in lung tissue were measured by immunohistochemistry and mucus secretion by histochemistry. KEY RESULTS: Preventive or therapeutic treatments with alpha-humulene, but not with trans-caryophyllene, significantly reduced the eosinophil recruitment to the BALF. In addition, alpha-humulene recovery INF-gamma and reduced the IL-5, CCL11 and LTB(4) levels in BALF, as well as the IL-5 production in mediastinal lymph nodes (in vitro assay). Furthermore, alpha-humulene decreased the NF-kB and the AP-1 activation, the expression of P-selectin and the increased mucus secretion in the lung. CONCLUSIONS AND IMPLICATIONS: alpha-Humulene, given either orally or by aerosol, exhibited marked anti-inflammatory properties in a murine model of airways allergic inflammation, an effect that seemed to be mediated via reduction of inflammatory mediators, adhesion molecule expression and transcription factors activation.

Anti-inflammatory properties of extracts, fractions and lignans isolated from Phyllanthus amarus.

This study assessed the anti-inflammatory effect of the extracts and purified lignans obtained from Phyllanthus amarus. Given orally, the hexane extract (HE), the lignan-rich fraction (LRF), or the lignans phyltetralin, nirtetralin, niranthin, but not hypophyllanthin or phyllanthin, inhibited carrageenan (Cg)-induced paw oedema and neutrophil influx. The HE, the LRF or nirtetralin also inhibited the increase of IL1-beta tissue levels induced by Cg. Furthermore, bradykinin (BK)-, platelet activating factor (PAF)- and endothelin-1 (ET-1)-induced paw oedema were significantly inhibited by the HE or LRF while histamine- and substance P-induced paw oedema were unaffected. Finally, nirtetralin or phyltetralin caused inhibition of paw oedema induced by PAF or ET-1. These results show that the HE, the LRF and the lignans niranthin, phyltetralin and nirtetralin exhibited marked anti-inflammatory properties and suggest that these lignans seem to be the main active principles responsible for the anti-inflammatory properties reported for the HE of P. amarus.