5-lipoxygenase- and Glucocorticoid-dependent eosinophilia in a novel surgical model in mice.

BACKGROUND: Subcutaneous implants of heat-coagulated egg white (egg white implants, EWI) induce intense local eosinophilia and prime for hyperreactivity following airway ovalbumin challenge. The roles of allergen sensitization, surgical trauma-induced glucocorticoids, and the 5-lipoxygenase (5-LO) pathway were hitherto unexplored in this model, in which quantitative recovery and large-scale purification of the eosinophils from the inflammatory site for functional and immunopharmacological studies are difficult to achieve. METHODS: We overcame this limitation by shifting the implantation site to the peritoneal cavity (EWIp), thereby enabling quantitative leukocyte retrieval. RESULTS: By day 7 post-surgery, eosinophil counts reached ~ 30% of all leukocytes recovered. Eosinophilia was prevented by: a) induction of allergen-specific oral tolerance to ovalbumin, the main allergen in egg white; b) inactivation of the 5-lipoxygenase pathway; c) blockade of endogenous glucocorticoid signaling by pretreatment with metirapone plus mifepristone before surgery. Highly purified eosinophils (~99% pure) could be obtained from the peritoneal exudate of EWIp-carrier mice in 2 simple, antibody-free steps. Preparative-scale yields, suitable for most biochemical, pharmacological, and molecular applications, were routinely obtained, and could be further enhanced through addition of pre-or post-surgery immunization steps (active or adoptive). The recovered eosinophils were fully functional in vivo, as demonstrated by the transfer of purified eosinophils into eosinophil-deficient Δdbl-GATA-1-KO mice, which upon subsequent challenge with eotaxin-1 present secondary accumulation of neutrophils, but not of mononuclear phagocytes. CONCLUSION: These findings document glucocorticoid-, allergen- and 5-lipoxygenase-dependent eosinophilia, which makes EWIp carriers an abundant source of pure, nontransgenic eosinophils for immunopharmacological studies.

Cutting Edge: Severe SARS-CoV-2 Infection in Humans Is Defined by a Shift in the Serum Lipidome, Resulting in Dysregulation of Eicosanoid Immune Mediators.

The COVID-19 pandemic has affected more than 20 million people worldwide, with mortality exceeding 800,000 patients. Risk factors associated with severe disease and mortality include advanced age, hypertension, diabetes, and obesity. Each of these risk factors pathologically disrupts the lipidome, including immunomodulatory eicosanoid and docosanoid lipid mediators (LMs). We hypothesized that dysregulation of LMs may be a defining feature of the severity of COVID-19. By examining LMs and polyunsaturated fatty acid precursor lipids in serum from hospitalized COVID-19 patients, we demonstrate that moderate and severe disease are separated by specific differences in abundance of immune-regulatory and proinflammatory LMs. This difference in LM balance corresponded with decreased LM products of ALOX12 and COX2 and an increase LMs products of ALOX5 and cytochrome p450. Given the important immune-regulatory role of LMs, these data provide mechanistic insight into an immuno-lipidomic imbalance in severe COVID-19.

Protective activities of distinct omega-3 enriched oils are linked to their ability to upregulate specialized pro-resolving mediators.

Clinical studies using a range of omega-3 supplements have yielded conflicting results on their efficacy to control inflammation. Omega-3 fatty acids are substrate for the formation of potent immune-protective mediators, termed as specialized pro-resolving mediators (SPM). Herein, we investigated whether observed differences in the potencies of distinct omega-3 supplements were linked with their ability to upregulate SPM formation. Using lipid mediator profiling we found that four commercially available supplements conferred a unique SPM signature profile to human macrophages, with the overall increases in SPM concentrations being different between the four supplements. These increases in SPM concentrations were linked with an upregulation of macrophage phagocytosis and a decreased uptake of oxidized low-density lipoproteins. Pharmacological inhibition of two key SPM biosynthetic enzymes 5-Lipoxygenase or 15-Lipoxygenase reversed the macrophage-directed actions of each of the omega-3 supplements. Furthermore, administration of the two supplements that most potently upregulated macrophage SPM formation and reprogrammed their responses in vitro, to APOE-/- mice fed a western diet, increased plasma SPM concentrations and reduced vascular inflammation. Together these findings support the utility of SPM as potential prognostic markers in determining the utility of a given supplement to regulate macrophage responses and inflammation.

Tuft-Cell-Derived Leukotrienes Drive Rapid Anti-helminth Immunity in the Small Intestine but Are Dispensable for Anti-protist Immunity.

Helminths, allergens, and certain protists induce type 2 immune responses, but the underlying mechanisms of immune activation remain poorly understood. In the small intestine, chemosensing by epithelial tuft cells results in the activation of group 2 innate lymphoid cells (ILC2s), which subsequently drive increased tuft cell frequency. This feedforward circuit is essential for intestinal remodeling and helminth clearance. ILC2 activation requires tuft-cell-derived interleukin-25 (IL-25), but whether additional signals regulate the circuit is unclear. Here, we show that tuft cells secrete cysteinyl leukotrienes (cysLTs) to rapidly activate type 2 immunity following chemosensing of helminth infection. CysLTs cooperate with IL-25 to activate ILC2s, and tuft-cell-specific ablation of leukotriene synthesis attenuates type 2 immunity and delays helminth clearance. Conversely, cysLTs are dispensable for the tuft cell response induced by intestinal protists. Our findings identify an additional tuft cell effector function and suggest context-specific regulation of tuft-ILC2 circuits within the small intestine.

AGER-Mediated Lipid Peroxidation Drives Caspase-11 Inflammasome Activation in Sepsis.

Inflammasome activation can trigger an inflammatory and innate immune response through the release of cytokines and induction of pyroptosis. A dysfunctional inflammasome has been implicated in the development of human pathologies, including sepsis and septic shock. Here, we show that advanced glycosylation end-product specific receptor (AGER/RAGE) is required for caspase-11 inflammasome activation in macrophages. A nuclear damage-associated molecular pattern (nDAMP) complex, including high-mobility group box 1, histone, and DNA, can promote caspase-11-mediated gasdermin D cleavage, interleukin 1ß proteolytic maturation, and lactate dehydrogenase release. The inhibition of AGER-mediated lipid peroxidation via arachidonate 5-lipoxygenase (ALOX5) limits caspase-11 inflammasome activation and pyroptosis in macrophages in response to nDAMPs or cytosolic lipopolysaccharide. Importantly, the pharmacologic inhibition of the AGER-ALOX5 pathway or global depletion (Ager-/-) or conditional depletion of AGER in myeloid cells (AgerMye-/-) protects against lipopolysaccharide-induced septic death in poly(I:C)-primed mice. These data identify a molecular basis for caspase-11 inflammasome activation and provide a potential strategy to treat sepsis.

Molecular characterization of big-belly seahorse (Hippocamus abdominalis) arachidonate 5-lipoxygenase (HaALOX5): First evidence of an immune defensive role by induced immunological stress in teleost.

Arachidonate 5-lipoxygenase (ALOX5) is an essential enzyme for the biosynthesis of leukotrienes, which are pro-inflammatory and anti-inflammatory mediators. In this study, the ALOX5 paralog of the big-belly seahorse (Hippocampus abdominalis; HaALOX5) was identified from our transcriptome database, and then molecularly and functionally characterized to determine its oxygenation capability and expression under pathogenic stress. The coding sequence of HaALOX5 consisted of 2025 bp and encoded a protein of 674 amino acids in length. Sequence and phylogenetic tree analysis of HaALOX5 revealed a close relationship with its corresponding teleost HaALOX5 counterparts. Structure prediction detected an N-terminal regulatory C2-like domain and a C-terminal catalytic domain, which are the two main functional domains in ALOX5 enzymes. Quantitative PCR showed that HaALOX5 was expressed in all the analyzed tissues at different magnitudes. The highest expression was detected in the intestine and stomach. In blood cells, the liver and the intestine, HaALOX5 transcripts were significantly elevated at many post injection time points, when immune challenged with lipopolysaccharide, polyinosinic:polycytidylic acid, and Streptococcus iniae, indicating its contribution to post immune defense mechanisms in the seahorse.

Trauma and hemorrhagic shock activate molecular association of 5-lipoxygenase and 5-lipoxygenase-Activating protein in lung tissue.

BACKGROUND: Post-traumatic lung injury following trauma and hemorrhagic shock (T/HS) is associated with significant morbidity. Leukotriene-induced inflammation has been implicated in the development of post-traumatic lung injury through a mechanism that is only partially understood. Postshock mesenteric lymph returning to the systemic circulation is rich in arachidonic acid, the substrate of 5-lipoxygenase (ALOX5). ALOX5 is the rate-limiting enzyme in leukotriene synthesis and, following T/HS, contributes to the development of lung dysfunction. ALOX5 colocalizes with its cofactor, 5-lipoxygenase-activating protein (ALOX5AP), which is thought to potentiate ALOX5 synthetic activity. We hypothesized that T/HS results in the molecular association and nuclear colocalization of ALOX5 and ALOX5AP, which ultimately increases leukotriene production and potentiates lung injury. MATERIALS AND METHODS: To examine these molecular interactions, a rat T/HS model was used. Post-T/HS tissue was evaluated for lung injury through both histologic analysis of lung sections and biochemical analysis of bronchoalveolar lavage fluid. Lung tissue was immunostained for ALOX5 and ALOX5AP with association and colocalization evaluated by fluorescence resonance energy transfer. In addition, rats undergoing T/HS were treated with MK-886, a known ALOX5AP inhibitor. RESULTS: ALOX5 levels increase and ALOX5/ALOX5AP association occurred after T/HS, as evidenced by increases in total tissue fluorescence and fluorescence resonance energy transfer signal intensity, respectively. These findings coincided with increased leukotriene production and with the histological changes characteristic of lung injury. ALOX5/ALOX5AP complex formation, leukotriene production, and lung injury were decreased after inhibition of ALOX5AP with MK-886. CONCLUSIONS: These results suggest that the association of ALOX5/ALOX5AP contributes to leukotriene-induced inflammation and predisposes the T/HS animal to lung injury.

Prominent release of lipoxygenase generated mediators in a murine house dust mite-induced asthma model.

The profile of activation of lipid mediator (LM) pathways in asthmatic airway inflammation remains unclear. This experimental study quantified metabolite levels of ω3-, ω6- and ω9-derived polyunsaturated fatty acids in bronchoalveolar lavage fluid (BALF) after 4-weeks of repeated house dust mite (HDM) exposure in a murine (C57BL/6) asthma model. The challenge induced airway hyperresponsiveness, pulmonary eosinophil infiltration, but with low and unchanged mast cell numbers. Of the 112 screened LMs, 26 were increased between 2 to >25-fold in BALF with HDM treatment (p < 0.05, false discovery rate = 5%). While cysteinyl-leukotrienes were the most abundant metabolites at baseline, their levels did not increase after HDM treatment, whereas elevation of PGD2, LTB4 and multiple 12/15-lipoxygenase products, such as 5,15-DiHETE, 15-HEDE and 15-HEPE were observed. We conclude that this model has identified a global lipoxygenase activation signature, not linked to mast cells, but with aspects that mimic chronic allergic airway inflammation in asthma.

5-/12-Lipoxygenase-linked cascade contributes to the IL-33-induced synthesis of IL-13 in mast cells, thus promoting asthma development.

BACKGROUND: As asthma progresses, the levels of IL-33 in serum are markedly increased and contribute to asthmatic development and exacerbation. Mast cells, one of the principal effector cells in the pathogenesis of asthma, express high levels of the IL-33 receptor ST2 and have been shown to be activated by IL-33. Thus, IL-33 stimulates mast cells to produce Th2-type cytokines such as IL-13, thus contributing to asthmatic development. However, the signaling mechanism for IL-33-induced synthesis of Th2 cytokines, particularly IL-13, has not been fully elucidated in mast cells. METHODS: The role of 5- or 12-LO in the IL-33-induced synthesis of IL-13 was investigated using knockdown or pharmacological inhibitors in bone marrow-derived mast cells (BMMCs) and animal model. RESULTS: Blockade of 5- or 12-LO significantly suppressed IL-33-induced synthesis of IL-13 in BMMCs. The subsequent action of 5- and 12-LO metabolites through their specific receptor, BLT2, was also critical for IL-33-induced synthesis of IL-13. We also demonstrated that the MyD88-p38 kinase cascade lies upstream of 5-/12-LO and that NF-κB lies downstream of 5-/12-LO to mediate the IL-33-induced synthesis of IL-13 in mast cells. Consistent with these findings, we observed that in an IL-33-administered asthmatic airway inflammation model, IL-13 levels were markedly increased in bronchoalveolar lavage fluid, but its levels were markedly suppressed by treatment with inhibitors of 5-LO, 12-LO or BLT2, further suggesting roles of 5-/12-LO in IL-33-induced IL-13 production. CONCLUSION: Our results suggest that "MyD88-5-/12-LO-BLT2-NF-κB" cascade significantly contributes to the IL-33-induced synthesis of IL-13 in mast cells, thus potentially contributing to asthmatic development and exacerbation.

The inhibition of 5-Lipoxygenase (5-LO) products leukotriene B4 (LTB4) and cysteinyl leukotrienes (cysLTs) modulates the inflammatory response and improves cutaneous wound healing.

To analyze the participation of the enzyme 5-lipoxygenase (5-LO) in skin repair, WT wounds were compared to those in 5-LO deficient mice (5-LO-/-), which presented faster closure and reduced inflammatory infiltrate in the skin, together with increased CD4 regulatory T cells markers in the draining lymph nodes. The 5-LO-/- wounds also had diminished TNF-α, CCL11, CCL7, CCL2, CXCL9, CCR1 and CXCR2 mRNA expression in the lesions, besides differential extracellular matrix remodeling. Furthermore, when cysteinyl leukotriene (cysLT) and leukotriene (LTB4) receptors were antagonized in WT mice, there was a remarkable reduction in TNF-α expression and faster skin healing, similarly to the findings in 5-LO-/- animals. Finally, our results suggested that 5-LO products, in special cysLT and LTB4, underline skin inflammation that follows skin injury and their neutralization may be an important strategy to improve cutaneous healing.

Recent advances in the search for novel 5-lipoxygenase inhibitors for the treatment of asthma.

The products of 5-lipoxygenase are synthesized and released in the airway when an asthmatic reaction occurs. 5-lipoxygenase via arachidonic acid metabolism produces leukotrienes that mediate bronchoconstriction and inflammatory modifications essential in the pathophysiology of asthma. Until to now, only one approved 5-LO inhibitor, zileuton, can be found as a potential therapy for asthma. With the increasing number of indications for anti-leukotriene (anti-LT) drugs, the development of 5-LO inhibitor agents becomes increasingly important. The present MiniReview reports an update on 5-LO inhibitors currently under clinical investigation. In addition, the latest advances focused on the development of new 5-lipoxygenase inhibitors as asthma anti-inflammatory agents are also discussed.

5-Lipoxygenase Knockout Aggravated Apical Periodontitis in a Murine Model.

5-Lipoxygenase (5-LO) plays a vital role in the host innate immune response, including bacteria-induced inflammation. Apical periodontitis (AP) is due to immune disorders caused by imbalances between bacterial invasion and subsequent host defense response. In this work, we investigated the role of 5-lipoxygenase in AP by using 5- lo knockout mice (5- lo-/- mice). Results showed that 5- lo-/- mice had greater periapical bone loss and more osteoclasts positive for tartrate-resistant acid phosphatase staining than did wild-type mice, as determined by micro-computed tomography and histologic staining. The inflammation- and osteoclastogenesis-related factors IL-1ß, TNF-α, RANK, and RANKL were also significantly elevated in 5- lo-/- mice, whereas osteoprotegerin was reduced. Furthermore, peritoneal macrophages from 5- lo-/- mice revealed an obviously impaired ability to phagocytose the AP pathogenic bacteria Fusobacterium nucleatum. In vivo experiments confirmed that 5- lo knockout led to decreased macrophage recruitment and increased F. nucleatum infection around the periapical area due to decreased leukotriene B4 and LXA4 production. All these results showed that 5- lo knockout impaired the host innate immune system to promote the release of bone resorption-related factors. Therefore, 5- lo deficiency aggravated AP in an experimental murine AP model.

Leukotriene signaling via ALOX5 and cysteinyl leukotriene receptor 1 is dispensable for in vitro growth of CD34+CD38- stem and progenitor cells in chronic myeloid leukemia.

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL+CD34+CD38- cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34+CD38- cells from 7 CML patients. The majority of the single leukemic BCR-ABL+CD34+CD38- cells expressed cysteinyl leukotriene receptors CYSLT1 and CYSLT2. However, montelukast, an inhibitor of CYSLT1, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLT1 signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients.

Loss of natural killer T cells promotes pancreatic cancer in LSL-KrasG12D/+ mice.

The role of the unique T-cell population, natural killer T (NKT) cells, which have similar functions to NK cells in pancreatic cancer (PC), is not yet evaluated. To address the regulatory roles of NKT cells on tumour progression through tumour-associated macrophages (TAM) and their production of microsomal prostaglandin E synthase-1 (mPGES-1) and 5-lipoxygenase (5-LOX) in (Kras)-driven pancreatic tumour (KPT) progression, we crossed CD1d-/- mice deficient in both invariant and variant NKT cells with the KrasG12D mice. Loss of NKT cells significantly increased pancreatic intraepithelial neoplasia (PanIN) lesions and also increased 5-LOX and mPGES-1 expression in M2-type macrophages and cancer stem-like cells in pancreatic tumours. Pharmacological inhibition of mPGES-1 and 5-LOX in M2 macrophages with specific inhibitor YS-121 in KPT-CD1d-/- mice decreased PanIN lesions and suppressed tumour growth in association with elevated levels of active CD8a cells. Hence, NKT cells regulate PC by modulating TAMs (M2) through mPGES-1 and 5-LOX; and the absence of NKT cells leads to aggressive development of PC.

Altering the Anti-inflammatory Lipoxin Microenvironment: a New Insight into Kaposi's Sarcoma-Associated Herpesvirus Pathogenesis.

Lipoxins are host anti-inflammatory molecules that play a vital role in restoring tissue homeostasis. The efficacy of lipoxins and their analog epilipoxins in treating inflammation and its associated diseases has been well documented. Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL) are two well-known inflammation related diseases caused by Kaposi's sarcoma-associated herpesvirus (KSHV). Controlling inflammation is one of the strategies adopted to treat KS and PEL, a primary motivation for exploring and evaluating the therapeutic potential of using lipoxins. This study documents how KSHV manipulates and downregulates the secretion of the anti-inflammatory lipoxin A4 in host cells and the viral factors involved in this process using in vitro KS and PEL cells as models. The presence of the lipoxin A4 receptor/formyl peptidyl receptor (ALX/FPR) in KS patient tissue sections and in vitro KS and PEL cell models offers a novel possibility for treating KS and PEL with lipoxins. Treating de novo KSHV-infected endothelial cells with lipoxin and epilipoxin creates an anti-inflammatory environment by decreasing the levels of NF-κB, AKT, ERK1/2, COX-2, and 5-lipoxygenase. Lipoxin treatment on CRISPR/CAS9 technology-mediated ALX/FPR gene deletion revealed the importance of the lipoxin receptor ALX for effective lipoxin signaling. A viral microRNA (miRNA) cluster was identified as the primary factor contributing to the downregulation of lipoxin A4 secretion in host cells. The KSHV miRNA cluster probably targets enzyme 15-lipoxygenase, which is involved in lipoxin A4 synthesis. This study provides a new insight into the potential treatment of KS and PEL using nature's own anti-inflammatory molecule, lipoxin. IMPORTANCE: KSHV infection has been shown to upregulate several host proinflammatory factors, which aid in its survival and pathogenesis. The influence of KSHV infection on anti-inflammatory molecules is not well studied. Since current treatment methods for KS and PEL are fraught with unwanted side effects and low efficiency, the search for new therapeutics is therefore imperative. The use of nature's own molecule lipoxin as a drug is promising. This study opens up new domains in KSHV research focusing on how the virus modulates lipoxin secretion and warrants further investigation of the therapeutic potential of lipoxin using in vitro cell models for KS and PEL.

Expression Profile of Cytokines and Enzymes mRNA in Blood Leukocytes of Dogs with Leptospirosis and Its Associated Pulmonary Hemorrhage Syndrome.

BACKGROUND: Dogs with leptospirosis show similar organ manifestations and disease course as human patients, including acute kidney injury and pulmonary hemorrhage, making this naturally-occurring infection a good animal model for human leptospirosis. Expression patterns of cytokines and enzymes have been correlated with disease manifestations and clinical outcome in humans and animals. The aim of this study was to describe mRNA expression of pro- and anti-inflammatory mediators in canine leptospirosis and to compare it with other renal diseases to identify patterns characterizing the disease and especially its pulmonary form. METHODOLOGY AND PRINCIPAL FINDINGS: The mRNA abundance of cytokines (IL-1α, IL-1ß, IL-8, IL-10, TNF-α, TGF-ß) and enzymes (5-LO, iNOS) was measured prospectively in blood leukocytes from 34 dogs with severe leptospirosis and acute kidney injury, including 22 dogs with leptospirosis-associated pulmonary hemorrhages. Dogs with leptospirosis were compared to 14 dogs with acute kidney injury of other origin than leptospirosis, 8 dogs with chronic kidney disease, and 10 healthy control dogs. Canine leptospirosis was characterized by high 5-LO and low TNF-α expression compared to other causes of acute kidney injury, although the decreased TNF-α expression was also seen in chronic kidney disease. Leptospirosis-associated pulmonary hemorrhage was not characterized by a specific pattern, with only mild changes noted, including increased IL-10 and decreased 5-LO expression on some days in affected dogs. Fatal outcome from pulmonary hemorrhages was associated with low TNF-α, high IL-1ß, and high iNOS expression, a pattern possibly expressed also in dogs with other forms of acute kidney injury. CONCLUSION: The patterns of cytokine and enzyme expression observed in the present study indicate a complex pro- and anti-inflammatory response to the infection with leptospires. The recognition of these signatures may be of diagnostic and prognostic relevance for affected individuals and they may indicate options for newer therapies targeting the identified pathways.

Sonic hedgehog-responsive lipoxygenases and cyclooxygenase-2 modulate Dectin-1-induced inflammatory cytokines.

Immune responses during fungal infections are predominately mediated by 5/15-lipoxygenases (LO)- or cyclooxygenase (COX)-2-catalysed bioactive eicosanoid metabolites like leukotrienes, lipoxins and prostaglandins. Although few host mediators of fungi-triggered eicosanoid production have been established, the molecular mechanism of expression and regulation of 5-LO, 15-LO and COX-2 are not well-defined. Here, we demonstrate that, macrophages infected with representative fungi Candida albicans, Aspergillus flavus or Aspergillus fumigatus or those treated with Curdlan, a selective agonist of pattern recognition receptor for fungi Dectin-1, displays increased expression of 5-LO, 15-LO and COX-2. Interestingly, Dectin-1-responsive Syk pathway activates mTOR-sonic hedgehog (SHH) signaling cascade to stimulate the expression of these lipid metabolizing enzymes. Loss-of-function analysis of the identified intermediaries indicates that while Syk-mTOR-SHH pathway-induced 5-LO and 15-LO suppressed the Dectin-1-responsive pro-inflammatory signature cytokines like TNF-α, IL-1ß and IL-12, Syk-mTOR-SHH-induced COX-2 positively regulated these cytokines. Dectin-1-stimulated IL-6, however, is dependent on 5-LO, 15-LO and COX-2 activity. Together, the current study establishes Dectin-1-arbitrated host mediators that direct the differential regulation of immune responses during fungal infections and thus are potential candidates of therapeutic intervention.

5-Lipoxygenase negatively regulates Th1 response during Brucella abortus infection in mice.

Brucella abortus is a Gram-negative bacterium that infects humans and cattle, causing a chronic inflammatory disease known as brucellosis. A Th1-mediated immune response plays a critical role in host control of this pathogen. Recent findings indicate contrasting roles for lipid mediators in host responses against infections. 5-Lipoxygenase (5-LO) is an enzyme required for the production of the lipid mediators leukotrienes and lipoxins. To determine the involvement of 5-LO in host responses to B. abortus infection, we intraperitoneally infected wild-type and 5-LO-deficient mice and evaluated the progression of infection and concomitant expression of immune mediators. Here, we demonstrate that B. abortus induced the upregulation of 5-LO mRNA in wild-type mice. Moreover, this pathogen upregulated the production of the lipid mediators leukotriene B4 and lipoxin A4 in a 5-LO-dependent manner. 5-LO-deficient mice displayed lower bacterial burdens in the spleen and liver and less severe liver pathology, demonstrating an enhanced resistance to infection. Host resistance paralleled an increased expression of the proinflammatory mediators interleukin-12 (IL-12), gamma interferon (IFN-γ), and inducible nitric oxide synthase (iNOS) during the course of infection. Moreover, we demonstrated that 5-LO downregulated the expression of IL-12 in macrophages during B. abortus infection. Our results suggest that 5-LO has a major involvement in B. abortus infection, by functioning as a negative regulator of the protective Th1 immune responses against this pathogen.

Cysteinyl leukotriene receptor-1 antagonists as modulators of innate immune cell function.

Cysteinyl leukotrienes (cysLTs) are produced predominantly by cells of the innate immune system, especially basophils, eosinophils, mast cells, and monocytes/macrophages. Notwithstanding potent bronchoconstrictor activity, cysLTs are also proinflammatory consequent to their autocrine and paracrine interactions with G-protein-coupled receptors expressed not only on the aforementioned cell types, but also on Th2 lymphocytes, as well as structural cells, and to a lesser extent neutrophils and CD8(+) cells. Recognition of the involvement of cysLTs in the immunopathogenesis of various types of acute and chronic inflammatory disorders, especially bronchial asthma, prompted the development of selective cysLT receptor-1 (cysLTR1) antagonists, specifically montelukast, pranlukast, and zafirlukast. More recently these agents have also been reported to possess secondary anti-inflammatory activities, distinct from cysLTR1 antagonism, which appear to be particularly effective in targeting neutrophils and monocytes/macrophages. Underlying mechanisms include interference with cyclic nucleotide phosphodiesterases, 5'-lipoxygenase, and the proinflammatory transcription factor, nuclear factor kappa B. These and other secondary anti-inflammatory mechanisms of the commonly used cysLTR1 antagonists are the major focus of the current review, which also includes a comparison of the anti-inflammatory effects of montelukast, pranlukast, and zafirlukast on human neutrophils in vitro, as well as an overview of both the current clinical applications of these agents and potential future applications based on preclinical and early clinical studies.

Leukotriene B4 modulates P2X7 receptor-mediated Leishmania amazonensis elimination in murine macrophages.

ATP is an important signaling molecule in the immune system, and it is able to bind the P2X7 purinergic receptor. Recently, our group showed that ATP-treated macrophages eliminate Leishmania amazonensis. It has been reported that leukotriene B4 (LTB4) reduces the parasitic load of infected macrophages. Additionally, it has been demonstrated that the P2X7 receptor can induce PLA2 activation and arachidonic acid mobilization. Based on these findings, we investigated whether LTB4 is produced upon P2X7 receptor activation and examined whether LTB4 modulates parasite elimination. Using macrophages lacking the P2X7 receptor, we observed that ATP was not able to reduce L. amazonensis load. This result suggests a role of the P2X7 purinergic receptor in parasite elimination. In addition, ATP was sufficient to induce LTB4 release from infected control macrophages but not from macrophages lacking the P2X7 receptor. Moreover, we found that ATP failed to decrease the parasitic load in 5-lipoxygenase (LO)-deficient macrophages. Treatment with the 5-LO inhibitor AA861 also impairs the ATP effect on parasitic loads. Furthermore, macrophages from 5-LO knockout mice eliminated L. amazonensis in the presence of exogenous LTB4, and macrophages obtained from P2X7 receptor knockout mice eliminated L. amazonensis when incubated with ionomycin. Finally, we demonstrated that in the presence of CP105696, an antagonist for LTB4 high-affinity receptor, ATP was not able to reduce parasitic load. These results indicate that P2X7 receptor activation leads to LTB4 formation, which is required for L. amazonensis elimination.