The identification of the endogenous ligands of natural killer T cells reveals the presence of mammalian α-linked glycosylceramides.

Glycosylceramides in mammalian species are thought to be present in the form of ß-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylceramide synthase, both ß-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.

TLR9-mediated dendritic cell activation uncovers mammalian ganglioside species with specific ceramide backbones that activate invariant natural killer T cells.

CD1d-restricted invariant natural killer T (iNKT) cells represent a heterogeneous population of lipid-reactive T cells that are involved in many immune responses, mediated through T-cell receptor (TCR)-dependent and/or independent activation. Although numerous microbial lipid antigens (Ags) have been identified, several lines of evidence have suggested the existence of relevant Ags of endogenous origin. However, the identification of their precise nature as well as the molecular mechanisms involved in their generation are still highly controversial and ill defined. Here, we identified two mammalian gangliosides-namely monosialoganglioside GM3 and disialoganglioside GD3-as endogenous activators for mouse iNKT cells. These glycosphingolipids are found in Toll-like receptor-stimulated dendritic cells (DC) as several species varying in their N-acyl fatty chain composition. Interestingly, their ability to activate iNKT cells is highly dependent on the ceramide backbone structure. Thus, both synthetic GM3 and GD3 comprising a d18:1-C24:1 ceramide backbone were able to activate iNKT cells in a CD1d-dependent manner. GM3 and GD3 are not directly recognized by the iNKT TCR and required the Ag presenting cell intracellular machinery to reveal their antigenicity. We propose a new concept in which iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced structural changes in CD1d-expressing cells. Moreover, these gangliosides conferred partial protection in the context of bacterial infection. Thus, this report identified new biologically relevant lipid self-Ags for iNKT cells.

Preclinical testing of a broad-spectrum antimicrobial endotracheal tube coated with an innate immune synthetic mimic.

BACKGROUND: Endotracheal tubes provide an abiotic surface on which bacteria and fungi form biofilms, and the release of endotoxins and planktonic organisms can cause damaging inflammation and infections. OBJECTIVES: Ceragenins are small molecule mimics of antimicrobial peptides with broad-spectrum antibacterial and antifungal activity, and a ceragenin may be used to provide antimicrobial protection to the abiotic surface of an endotracheal tube. METHODS: A hydrogel film, containing CSA-131, was generated on endotracheal tubes. Elution of CSA-131 was quantified in drip-flow and static systems, antifungal and antibacterial activity was measured with repeated inoculation in growth media, biofilm formation was observed through electron microscopy, safety was determined by intubation of pigs with coated and uncoated endotracheal tubes. RESULTS: Optimized coatings containing CSA-131 provided controlled elution of CSA-131, with concentrations released of less than 1 µg/mL. The eluting ceragenin prevented fungal and bacterial colonization of coated endotracheal tubes for extended periods, while uncoated tubes were colonized by bacteria and fungi. Coated tubes were well tolerated in intubated pigs. CONCLUSIONS: Thin films containing CSA-131 provide protection against microbial colonization of endotracheal tubes. This protection prevents fungal and bacterial biofilm formation on the tubes and reduces endotoxin associated with tubes. This coating is well suited for decreasing the adverse effects of intubation associated with infection and inflammation.

Ceragenins are active against drug-resistant Candida auris clinical isolates in planktonic and biofilm forms.

Background: Candida auris has emerged as a serious threat to human health. Of particular concern are the resistance profiles of many clinical isolates, with some being resistant to multiple classes of antifungals. Objectives: Measure susceptibilities of C. auris isolates, in planktonic and biofilm forms, to ceragenins (CSAs). Determine the effectiveness of selected ceragenins in gel and cream formulations in eradicating fungal infections in tissue explants. Materials and methods: A collection of 100 C. auris isolates available at CDC was screened for susceptibility to a lead ceragenin. A smaller collection was used to characterize antifungal activities of other ceragenins against organisms in planktonic and biofilm forms. Effects of ceragenins on fungal cells and biofilms were observed via microscopy. An ex vivo model of mucosal fungal infection was used to evaluate formulated forms of lead ceragenins. Results: Lead ceragenins displayed activities comparable to those of known antifungal agents against C. auris isolates with MICs of 0.5-8 mg/L and minimum fungicidal concentrations (MFCs) of 2-64 mg/L. No cross-resistance with other antifungals was observed. Fungal cell morphology was altered in response to ceragenin treatment. Ceragenins exhibited activity against sessile organisms in biofilms. Gel and cream formulations including 2% CSA-44 or CSA-131 resulted in reductions of over 4 logs against established fungal infections in ex vivo mucosal tissues. Conclusions: Ceragenins demonstrated activity against C. auris, suggesting that these compounds warrant further study to determine whether they can be used for topical applications to skin and mucosal tissues for treatment of infections with C. auris and other fungi.

Discrete TCR Binding Kinetics Control Invariant NKT Cell Selection and Central Priming.

Invariant NKT (iNKT) cells develop and differentiate in the thymus, segregating into iNKT1/2/17 subsets akin to Th1/2/17 classical CD4+ T cells; however, iNKT TCRs recognize Ags in a fundamentally different way. How the biophysical parameters of iNKT TCRs influence signal strength in vivo and how such signals affect the development and differentiation of these cells are unknown. In this study, we manipulated TCRs in vivo to generate clonotypic iNKT cells using TCR retrogenic chimeras. We report that the biophysical properties of CD1d-lipid-TCR interactions differentially impacted the development and effector differentiation of iNKT cells. Whereas selection efficiency strongly correlated with TCR avidity, TCR signaling, cell-cell conjugate formation, and iNKT effector differentiation correlated with the half-life of CD1d-lipid-TCR interactions. TCR binding properties, however, did not modulate Ag-induced iNKT cytokine production. Our work establishes that discrete TCR interaction kinetics influence iNKT cell development and central priming.

Antibacterial and Antifungal Activities of Poloxamer Micelles Containing Ceragenin CSA-131 on Ciliated Tissues.

Ceragenins were designed as non-peptide mimics of endogenous antimicrobial peptides, and they display broad-spectrum antibacterial and antifungal activities, including the ability to eradicate established biofilms. These features of ceragenins make them attractive potential therapeutics for persistent infections in the lung, including those associated with cystic fibrosis. A characteristic of an optimal therapeutic for use in the lungs and trachea is the exertion of potent antimicrobial activities without damaging the cilia that play a critical role in these tissues. In previous work, potent antimicrobial activities of ceragenin CSA-131 have been reported; however, we found in ex vivo studies that this ceragenin, at concentrations necessary to eradicate established biofilms, also causes loss of cilia function. By formulating CSA-131 in poloxamer micelles, cilia damage was eliminated and antimicrobial activity was unaffected. The ability of CSA-131, formulated with a poloxamer, to reduce the populations of fungal pathogens in tracheal and lung tissue was also observed in ex vivo studies. These findings suggest that CSA-131, formulated in micelles, may act as a potential therapeutic for polymicrobial and biofilm-related infections in the lung and trachea.

Natural killer T (NKT)-B-cell interactions promote prolonged antibody responses and long-term memory to pneumococcal capsular polysaccharides.

Innate-like natural killer T (NKT) cells critically enhance cell and humoral immunity against infections through recognition of conserved microbial lipid antigens presented by CD1d-expressing antigen-presenting cells, and provision of CD40L and cytokine signals. Whereas NKT cells efficiently licensed dendritic cells to prime potent effector and memory T cells, studies based on model antigens such as alphagalactosylceramide-nitrophenyl conjugates concluded that help to B cells was associated with NKT follicular helper differentiation, but limited to short-term responses without induction of memory. We revisited this surprising conclusion in the context of the extracellular encapsulated pathogen Streptococcus pneumoniae, where recognition of lipid and capsular polysaccharide antigens by NKT cells and B cells, respectively, provide critical host protection. Using liposomal nanoparticles displaying synthetic lipid and polysaccharide antigens to elicit pure and direct NKT-B-cell interactions in vivo, we observed intense and prolonged antibody responses with isotype switch, affinity maturation, and long-lasting B-cell memory, despite modest or absent NKT follicular helper differentiation. Furthermore, conditional ablation of Cd1d demonstrated a requirement for a two-step process involving first cognate interactions with dendritic cells, for NKT cell activation, and then with B cells, for induction of isotype switch and memory. Thus, NKT help to B cells represents both a major arm of antimicrobial defense and a promising target for B-cell vaccines.

Impact of sugar stereochemistry on natural killer T cell stimulation by bacterial glycolipids.

Natural killer T (NKT) cells recognize glycolipids produced by Sphingomonas bacteria, and these glycolipids contain C6-oxidized sugars, either glucuronic acid or galacturonic acid, linked to ceramides. Glycolipids with gluco stereochemistry are the most prevalent. Multiple studies have demonstrated that galactosylceramides are more potent stimulators of NKT cells than their glucose isomers. To determine if this stereoselectivity is retained in the context of the C6-oxidized sugars found in bacterial glycolipids, we prepared two sets of gluco and galacto-glycolipids oxidized at their C6 positions and compared their NKT stimulatory properties. In the context of carboxylic acid groups at C6, gluco stereochemistry gave the more potent responses. We also prepared bacterial glycolipids containing more complex ceramide groups to determine if these chains impact NKT cell responses.

Synthesis of the pentasaccharide repeating unit from Ruminococcus gnavus and measurement of its inflammatory properties.

The roles played by the gut microbiome in human health are increasingly recognized, and the prevalence of specific microorganisms has been correlated with different diseases. For example, blooms of the Gram-positive bacterium Ruminococcus gnavus have been correlated with inflammatory bowel disease, and recently a polysaccharide produced by this organism was shown to stimulate release of inflammatory cytokines. This stimulation was proposed to signal through toll-like receptor 4 (TLR4). We have synthesized the pentasaccharide repeating unit of this polysaccharide and showed that it stimulates TNF-α and IL-6 release from bone marrow-derived dendritic cells (BMDCs) in a TLR4-dependent manner. A related glycan does not stimulate significant cytokine release, demonstrating TLR4 selectivity in glycan recognition.

Unravelling the structural complexity of glycolipids with cryogenic infrared spectroscopy.

Glycolipids are complex glycoconjugates composed of a glycan headgroup and a lipid moiety. Their modular biosynthesis creates a vast amount of diverse and often isomeric structures, which fulfill highly specific biological functions. To date, no gold-standard analytical technique can provide a comprehensive structural elucidation of complex glycolipids, and insufficient tools for isomer distinction can lead to wrong assignments. Herein we use cryogenic gas-phase infrared spectroscopy to systematically investigate different kinds of isomerism in immunologically relevant glycolipids. We show that all structural features, including isomeric glycan headgroups, anomeric configurations and different lipid moieties, can be unambiguously resolved by diagnostic spectroscopic fingerprints in a narrow spectral range. The results allow for the characterization of isomeric glycolipid mixtures and biological applications.

Translation of ceragenin affinity for bacteria to an imaging reagent for infection.

Responses to bacterial infections may be manifest systemically without evidence of the location of the infection site. A rapid means of pinpointing infection sites would be useful in providing effective and possibly localized treatment. Successful means of identifying infection sites would require two components: (1) a molecule capable of recognizing bacteria and (2) a means of communicating recognition. For the recognition element, we used a ceragenin, a small molecule with affinity for bacterial membranes that was designed as a mimic of endogenous antimicrobial peptides. For the communication element, we used 64Cu, which is a positron emitter. By conjugating a copper chelating group to the ceragenin, the two elements were combined. Chelation of 64Cu by the conjugate was effective and provided a stable complex that allowed in vivo imaging. When administered to mice in a thigh infection model, the 64Cu-labeled conjugate accumulated at the site of infection (right thigh) without accumulation at the complementary site (left thigh). This conjugate may provide a means of identifying infection sites in patients presenting general signs of infection without localized symptoms.

Proteomic Analysis of Resistance of Gram-Negative Bacteria to Chlorhexidine and Impacts on Susceptibility to Colistin, Antimicrobial Peptides, and Ceragenins.

Use of chlorhexidine in clinical settings has led to concerns that repeated exposure of bacteria to sub-lethal doses of chlorhexidine might result in chlorhexidine resistance and cross resistance with other cationic antimicrobials including colistin, endogenous antimicrobial peptides (AMPs) and their mimics, ceragenins. We have previously shown that colistin-resistant Gram-negative bacteria remain susceptible to AMPs and ceragenins. Here, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins by serial exposure of standard strains of Gram-negative bacteria to chlorhexidine to generate resistant populations of organisms. Furthermore, we performed a proteomics study on the chlorhexidine-resistant strains and compared them to the wild-type strains to find the pathways by which bacteria develop resistance to chlorhexidine. Serial exposure of Gram-negative bacteria to chlorhexidine resulted in four- to eight-fold increases in minimum inhibitory concentrations (MICs). Chlorhexidine-resistant organisms showed decreased susceptibility to colistin (8- to 32-fold increases in MICs) despite not being exposed to colistin. In contrast, chlorhexidine-resistant organisms had the same MICs as the original strains when tested with representative AMPs (LL-37 and magainin I) and ceragenins (CSA-44 and CSA-131). These results imply that there may be a connection between the emergence of highly colistin-resistant Gram-negative pathogens and the prevalence of chlorhexidine usage. Yet, use of chlorhexidine may not impact innate immune defenses (e.g., AMPs) and their mimics (e.g., ceragenins). Here, we also show that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa. Additionally, resistance to chlorhexidine resulted in elevated expression levels of proteins associated with chaperones, efflux pumps, flagella and cell metabolism. This study provides a comprehensive overview of the evolutionary proteomic changes in P. aeruginosa following exposure to chlorhexidine and colistin. These results have important clinical implications considering the continuous application of chlorhexidine in hospitals that could influence the emergence of colistin-resistant strains.

Lipid Antigen Presentation by CD1b and CD1d in Lysosomal Storage Disease Patients.

The lysosome has a key role in the presentation of lipid antigens by CD1 molecules. While defects in lipid antigen presentation and in invariant Natural Killer T (iNKT) cell response were detected in several mouse models of lysosomal storage diseases (LSD), the impact of lysosomal engorgement in human lipid antigen presentation is poorly characterized. Here, we analyzed the capacity of monocyte-derived dendritic cells (Mo-DCs) from Fabry, Gaucher, Niemann Pick type C and Mucopolysaccharidosis type VI disease patients to present exogenous antigens to lipid-specific T cells. The CD1b- and CD1d-restricted presentation of lipid antigens by Mo-DCs revealed an ability of LSD patients to induce CD1-restricted T cell responses within the control range. Similarly, freshly isolated monocytes from Fabry and Gaucher disease patients had a normal ability to present α-Galactosylceramide (α-GalCer) antigen by CD1d. Gaucher disease patients' monocytes had an increased capacity to present α-Gal-(1-2)-αGalCer, an antigen that needs internalization and processing to become antigenic. In summary, our results show that Fabry, Gaucher, Niemann Pick type C, and Mucopolysaccharidosis type VI disease patients do not present a decreased capacity to present CD1d-restricted lipid antigens. These observations are in contrast to what was observed in mouse models of LSD. The percentage of total iNKT cells in the peripheral blood of these patients is also similar to control individuals. In addition, we show that the presentation of exogenous lipids that directly bind CD1b, the human CD1 isoform with an intracellular trafficking to the lysosome, is normal in these patients.

Synthesis of diglycosylceramides and evaluation of their iNKT cell stimulatory properties.

Stimulation of iNKT cells is highly dependent on the structures of the glycolipids presented by CD1d. Furthermore, antigen processing and CD1d loading in lysosomes play central roles in controlling the stimulatory properties of glycolipid antigens. Previously, we determined that substitution at C6'' on alpha-galactosylceramides did not significantly impact stimulatory properties; however, it was not known if substitution at this position influenced lysosomal processing of oligoglycosylceramides. We have prepared a series of mono- and di-galactosylceramides to observe the impact of C6'' substitution on glycosidase truncation of these glycolipids. We found that substitution did not significantly impact glycosidase activity or loading into CD1d.

Susceptibility of Multidrug-Resistant Bacteria, Isolated from Water and Plants in Nigeria, to Ceragenins.

The continuous emergence of multidrug resistant pathogens is a major global health concern. Although antimicrobial peptides (AMPs) have shown promise as a possible means of combatting multidrug resistant strains without readily engendering resistance, costs of production and targeting by proteases limit their utility. Ceragenins are non-peptide AMP mimics that overcome these shortcomings while retaining broad-spectrum antimicrobial activity. To further characterize the antibacterial activities of ceragenins, their activities against a collection of environmental isolates of bacteria were determined. These isolates were isolated in Nigeria from plants and water. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of selected ceragenins and currently available antimicrobials against these isolates were measured to determine resistance patterns. Using scanning electron microscopy (SEM), we examined the morphological changes in bacterial membranes following treatment with ceragenins. Finally, we investigated the effectiveness of ceragenins in inhibiting biofilm formation and destroying established biofilms. We found that, despite high resistance to many currently available antimicrobials, including colistin, environmental isolates in planktonic and biofilm forms remain susceptible to ceragenins. Additionally, SEM and confocal images of ceragenin-treated cells confirmed the effective antibacterial and antibiofilm activity of ceragenins.

T cells control the generation of nanomolar-affinity anti-glycan antibodies.

Vaccines targeting glycan structures at the surface of pathogenic microbes must overcome the inherent T cell-independent nature of immune responses against glycans. Carbohydrate conjugate vaccines achieve this by coupling bacterial polysaccharides to a carrier protein that recruits heterologous CD4 T cells to help B cell maturation. Yet they most often produce low- to medium-affinity immune responses of limited duration in immunologically fit individuals and disappointing results in the elderly and immunocompromised patients. Here, we hypothesized that these limitations result from suboptimal T cell help. To produce the next generation of more efficacious conjugate vaccines, we have explored a synthetic design aimed at focusing both B cell and T cell recognition to a single short glycan displayed at the surface of a virus-like particle. We tested and established the proof of concept of this approach for 2 serotypes of Streptococcus pneumoniae. In both cases, these vaccines elicited serotype-specific, protective, and long-lasting IgG antibodies of nanomolar affinity against the target glycans in mice. We further identified a requirement for CD4 T cells in the anti-glycan antibody response. Our findings establish the design principles for improved glycan conjugate vaccines. We surmise that the same approach can be used for any microbial glycan of interest.

Improved proliferation of antigen-specific cytolytic T lymphocytes using a multimodal nanovaccine.

The present study investigated the immunoenhancing property of our newly designed nanovaccine, that is, its ability to induce antigen-specific immunity. This study also evaluated the synergistic effect of a novel compound PBS-44, an α-galactosylceramide analog, in boosting the immune response induced by our nanovaccine. The nanovaccine was prepared by encapsulating ovalbumin (ova) and an adjuvant within the poly(lactic-co-glycolic acid) nanoparticles. Quantitative analysis of our study data showed that the encapsulated vaccine was physically and biologically stable; the core content of our nanovaccine was found to be released steadily and slowly, and nearly 90% of the core content was slowly released over the course of 25 days. The in vivo immunization studies exhibited that the nanovaccine induced stronger and longer immune responses compared to its soluble counterpart. Similarly, intranasal inhalation of the nanovaccine induced more robust antigen-specific CD8+ T cell response than intraperitoneal injection of nanovaccine.

Efficacy of ABX196, a new NKT agonist, in prophylactic human vaccination.

We have assessed the immune-regulatory and adjuvant activities of a synthetic glycolipid, ABX196, a novel analog of the parental compound α-GalCer. As expected, ABX196 demonstrated a measurable and significant adjuvant effect in mice and monkeys with no appreciable toxicity at the doses used to promote immune responses. We performed a phase I/II dose escalation study of ABX196 in healthy volunteers, with the objectives to evaluate its safety profile, as well as its ability to be utilized as an adjuvant in the context of a prophylactic vaccine against hepatitis B. ABX196 was administered at three doses: 0.2, 0.4, and 2.0µg, in 44 subjects. In all the individuals injected with ABX196, peripheral blood NKT cells displayed hallmarks of activation, and 45% of them had measurable circulating IFN-γ 24h after the first administration. More importantly, the addition of ABX196 to the very poorly immunogenic HBs antigen resulted in protective anti-HBs antibody responses in majority of patients, demonstrating the adjuvant properties of ABX196 in human. Further analysis of the cohort of subjects receiving ABX196 with HBs antigen also indicates that a single injection appears sufficient to provide protection. A limited set of adverse events linked to the systemic delivery of ABX196 and access to the liver, is discussed in the context of formulation and the need to limit transport of ABX196 to secondary lymphoid tissues for maximal efficacy (Eudra-CT 2012-001566-15).

Synthesis of fungal glycolipid asperamide B and investigation of its ability to stimulate natural killer T cells.

The relationship between mold and asthma has been recognized for decades, but the molecular triggers of asthma generated by molds have not been fully elucidated. A glycolipid generated by Aspergillus species has recently been identified that triggers airway hyperreactivity via natural killer T cell activation. The synthesis of this glycolipid and structural variants designed to allow identification of the features of this glycolipid required for recognition by natural killer T cells is described.