Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis

Background

Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects.

Methods

The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models.

Results

We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation.

Conclusions

The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection.

General significance

Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.     

Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte–macrophage interaction

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis.Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1.In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.     

De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes

De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.     

A sphingolipid synthesis‐related protein OrmA in Aspergillus fumigatus is responsible for azole susceptibility and virulence

Previous studies identified that the budding yeast Saccharomyces cerevisiae have two sphingolipid synthesis‐related proteins, Orm1p and Orm2p, that negatively regulate the activities of SPT, which is a key rate‐limiting enzyme in sphingolipid synthesis. However, little is known about whether sphingolipids in the cell membrane, which are closely related to ergosterols, could affect the efficacy of azole drugs, which target to the ergosterol biosynthesis. In this study, through genome‐wide homologue search analysis, we found that the Aspergillus fumigatus genome only contains one Orm homologue, referred to as OrmA for which the protein expression could be induced by azole antifungals in a dose‐dependent manner. Deletion of ormA caused hypersensitivity to azoles, and adding the sphingolipid synthesis inhibitor myriocin rescued the azole susceptibility induced by lack of ormA. In contrast, overexpression of OrmA resulted in azole resistance, indicating that OrmA is a positive azole‐response regulator. Further mechanism analysis verified that OrmA is related to drug susceptibility by affecting endoplasmic reticulum stress responses in an unfolded protein response pathway‐HacA‐dependent manner. Lack of ormA led to an abnormal profile of sphingolipid ceramide components accompanied by hypersensitivity to low temperatures. Furthermore, deletion of OrmA significantly reduced virulence in an immunosuppressed mouse model. The findings in this study collectively suggest that the sphingolipid metabolism pathway in A. fumigatus plays a critical role in azole susceptibility and fungal virulence.     

Fungal Pathogens in CF Airways: Leave or Treat?

Chronic airway infection plays an essential role in the progress of cystic fibrosis (CF) lung disease. In the past decades, mainly bacterial pathogens, such as Pseudomonas aeruginosa, have been the focus of researchers and clinicians. However, fungi are frequently detected in CF airways and there is an increasing body of evidence that fungal pathogens might play a role in CF lung disease. Several studies have shown an association of fungi, particularly Aspergillus fumigatus and Candida albicans, with the course of lung disease in CF patients. Mechanistically, in vitro and in vivo studies suggest that an impaired immune response to fungal pathogens in CF airways renders them more susceptible to fungi. However, it remains elusive whether fungi are actively involved in CF lung disease pathologies or whether they rather reflect a dysregulated airway colonization and act as microbial bystanders. A key issue for dissecting the role of fungi in CF lung disease is the distinction of dynamic fungal–host interaction entities, namely colonization, sensitization or infection. This review summarizes key findings on pathophysiological mechanisms and the clinical impact of fungi in CF lung disease.     

A novel role for sphingolipid intermediates in activation-induced cell death in T cells

Activation-induced cell death (AICD), a process mediated by CD95 and CD95 ligand (CD95L), plays a critical role in regulating homeostasis of the immune system. Although the role of sphingolipids such as ceramides has been suggested to participate in CD95-mediated apoptosis, the exact role of these molecules in this process remains controversial. We employed myriocin, a specific inhibitor of serine palmitoyl-CoA transferase that mediates the first commitment step in sphingolipid synthesis. We found that myriocin could effectively block AICD in T-cell hybridomas and T-cell blasts. However, fumonisin B1, an inhibitor of the final step of ceramide synthesis, or inhibitors of sphingomyelinases did not prevent AICD. Furthermore, ceramide analogues, such as C2 and C6, could not reverse the inhibitory effect of myriocin. Interestingly, sphinganine, an intermediate of ceramide synthesis, completely reversed the inhibitory effect of myriocin, indicating a critical role of sphinganine. Myriocin did not modulate the expression of CD95 or CD95L, instead, it interfered with the early steps of CD95-mediated caspase activation. Therefore, we have uncovered a novel mechanism by which sphingolipid intermediates regulate CD95-mediated apoptosis.     

IL-1α Signaling Is Critical for Leukocyte Recruitment after Pulmonary Aspergillus fumigatus Challenge

Aspergillus fumigatus is a mold that causes severe pulmonary infections. Our knowledge of how A. fumigatus growth is controlled in the respiratory tract is developing, but still limited. Alveolar macrophages, lung resident macrophages, and airway epithelial cells constitute the first lines of defense against inhaled A. fumigatus conidia. Subsequently, neutrophils and inflammatory CCR2+ monocytes are recruited to the respiratory tract to prevent fungal growth. However, the mechanism of neutrophil and macrophage recruitment to the respiratory tract after A. fumigatus exposure remains an area of ongoing investigation. Here we show that A. fumigatus pulmonary challenge induces expression of the inflammasome-dependent cytokines IL-1β and IL-18 within the first 12 hours, while IL-1α expression continually increases over at least the first 48 hours. Strikingly, Il1r1-deficient mice are highly susceptible to pulmonary A. fumigatus challenge exemplified by robust fungal proliferation in the lung parenchyma. Enhanced susceptibility of Il1r1-deficient mice correlated with defects in leukocyte recruitment and anti-fungal activity. Importantly, IL-1α rather than IL-1β was crucial for optimal leukocyte recruitment. IL-1α signaling enhanced the production of CXCL1. Moreover, CCR2+ monocytes are required for optimal early IL-1α and CXCL1 expression in the lungs, as selective depletion of these cells resulted in their diminished expression, which in turn regulated the early accumulation of neutrophils in the lung after A. fumigatus challenge. Enhancement of pulmonary neutrophil recruitment and anti-fungal activity by CXCL1 treatment could limit fungal growth in the absence of IL-1α signaling. In contrast to the role of IL-1α in neutrophil recruitment, the inflammasome and IL-1β were only essential for optimal activation of anti-fungal activity of macrophages. As such, Pycard-deficient mice are mildly susceptible to A. fumigatus infection. Taken together, our data reveal central, non-redundant roles for IL-1α and IL-1β in controlling A. fumigatus infection in the murine lung.     

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G(2) /M phase arrest

Objectives: Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti‐cancer treatment have been sought from natural resources. Here, we have investigated anti‐proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the de novo sphingolipid pathway, and its mechanism in B16F10 melanoma cells. Material and methods: We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate levels were analysed by HPLC. Results: Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G₂/M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21^waf1/cip1 was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate in myriocin‐treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells. Conclusions: Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21^waf1/cip1, followed by inhibition of cyclin B1 and cdc2, resulting in G₂/M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism‐based therapy for this type of skin cancer.     

Pharmacokinetics of Azole Antifungals in Cystic Fibrosis

Defects in mucociliary clearance predispose cystic fibrosis (CF) patients to airway colonization and infection by various fungi, especially Aspergillus fumigatus. Although the clinical significance of airway fungal colonization is not clear, several studies have suggested its association with worsening lung function and increased risk of CF exacerbations. Antifungal triazole agents have been used in CF patients with airway fungal colonization or infections with varying results. Limited pharmacokinetic studies to date have demonstrated high inter-subject variability of triazole levels among CF patients. This review discusses the basic principles of pharmacokinetics, the pharmacokinetic changes associated with CF and the effect of CF on the pharmacokinetic principles of azole antifungals. The inconsistent azole serum levels in CF patients may be associated with sub-therapeutic (thus risk of therapeutic failure and/or emergence of azole-resistant fungi) or supratherapeutic exposures (thus potential risk of azole toxicity), suggesting that therapeutic dose monitoring is necessary in CF patients.     

Elimination of Aspergillus fumigatus conidia from the airways of mice with allergic airway inflammation

Background

Aspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores.

Methods

Using BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G⁺ neutrophils and MHC II⁺ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways.

Results

Allergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application.

Conclusions

Aspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.     

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.     

Myriocin, an inhibitor of serine palmitoyl transferase, impairs the uptake of transferrin and low-density lipoprotein in mammalian cells

The role of sphingolipids in clathrin-mediated endocytosis is only poorly understood in mammalian cells. Thus the relationship between sphingolipid de novo synthesis and clathrin-mediated endocytosis of transferrin were studied in L929 fibroblasts and two other cell lines. Endocytosis was measured using live cell imaging with fluorescent transferrin or (125)I-transferrin. Lipids were primarily measured using electrospray ionization tandem mass spectrometry. At physiological temperature, transferrin uptake was significantly decreased by the inhibitor of serine palmitoyl transferase myriocin. Myriocin inhibited also the uptake of low-density lipoproteins. The endocytosis inhibition by myriocin could be released by the addition of sphingoid base and by the protein phosphorylation effectors phorbol-12-myristate, 13-acetate (PMA) and okadaic acid. Myriocin influenced not only sphingolipids but also the glycerophospholipid profile. The study of phosphatidylcholine species shows adaptations to more saturated, alkylated and longer fatty acid moieties. The reported results imply that in mammalian cells, at 37°C, sphingolipid de novo synthesis is required for clathrin-mediated endocytosis.     

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2^-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2^-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2^-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.     

A Murine Inhalation Model to Characterize Pulmonary Exposure to Dry Aspergillus fumigatus Conidia

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×10⁵ twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8⁺ IL17⁺ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.     

Aspergillus antigen induces robust Th2 cytokine production,inflammation, airway hyperreactivity and fibrosis in the absence of MCP-1 or CCR2

BackgroundAsthma is characterized by type 2 T-helper cell (Th2) inflammation, goblet cell hyperplasia, airway hyperreactivity, and airway fibrosis. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and its receptor, CCR2, have been shown to play important roles in the development of Th2 inflammation. CCR2-deficient mice have been found to have altered inflammatory and physiologic responses in some models of experimental allergic asthma, but the role of CCR2 in contributing to inflammation and airway hyperreactivity appears to vary considerably between models. Furthermore, MCP-1-deficient mice have not previously been studied in models of experimental allergic asthma.MethodsTo test whether MCP-1 and CCR2 are each required for the development of experimental allergic asthma, we applied an Aspergillus antigen-induced model of Th2 cytokine-driven allergic asthma associated with airway fibrosis to mice deficient in either MCP-1 or CCR2. Previous studies with live Aspergillus conidia instilled into the lung revealed that MCP-1 and CCR2 play a role in anti-fungal responses; in contrast, we used a non-viable Aspergillus antigen preparation known to induce a robust eosinophilic inflammatory response.ResultsWe found that wild-type C57BL/6 mice developed eosinophilic airway inflammation, goblet cell hyperplasia, airway hyperreactivity, elevations in serum IgE, and airway fibrosis in response to airway challenge with Aspergillus antigen. Surprisingly, mice deficient in either MCP-1 or CCR2 had responses to Aspergillus antigen similar to those seen in wild-type mice, including production of Th2 cytokines.ConclusionWe conclude that robust Th2-mediated lung pathology can occur even in the complete absence of MCP-1 or CCR2.     

Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge

Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection.