Spatially Resolved Molecular Analysis of Host Response to Medical Device Implantation Using the 3D OrbiSIMS Highlights a Critical Role for Lipids.

A key goal for implanted medical devices is that they do not elicit a detrimental immune response. Macrophages play critical roles in the modulation of the host immune response and are the cells responsible for persistent inflammatory reactions to implanted biomaterials. Two novel immune-instructive polymers that stimulate pro- or anti-inflammatory responses from macrophages in vitro are investigated. These also modulate in vivo foreign body responses (FBR) when implanted subcutaneously in mice. Immunofluorescent staining of tissue abutting the polymer reveals responses consistent with pro- or anti-inflammatory responses previously described for these polymers. Three Dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) analysis to spatially characterize the metabolites in the tissue surrounding the implant, providing molecular histology insight into the metabolite response in the host is applied. For the pro-inflammatory polymer, monoacylglycerols (MG) and diacylglycerols (DG) are observed at increased intensity, while for the anti-inflammatory coating, the number of phospholipid species detected decreased, and pyridine and pyrimidine levels are elevated. Small molecule signatures from single-cell studies of M2 macrophages in vitro correlate with the in vivo observations, suggesting potential for prediction. Metabolite characterization by the 3D OrbiSIMS is shown to provide insight into the mechanism of bio-instructive materials as medical devices and to inform on the FBR to biomaterials.

Label-free Chemical Characterization of Polarized Immune Cells in vitro and Host Response to Implanted Bio-instructive Polymers in vivo Using 3D OrbiSIMS.

The Three-dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) is a secondary ion mass spectrometry instrument, a combination of a Time of Flight (ToF) instrument with an Orbitrap analyzer. The 3D OrbiSIMS technique is a powerful tool for metabolic profiling in biological samples. This can be achieved at subcellular spatial resolution, high sensitivity, and high mass-resolving power coupled with MS/MS analysis. Characterizing the metabolic signature of macrophage subsets within tissue sections offers great potential to understand the response of the human immune system to implanted biomaterials. Here, we describe a protocol for direct analysis of individual cells after in vitro differentiation of naïve monocytes into M1 and M2 phenotypes using cytokines. As a first step in vivo, we investigate explanted silicon catheter sections as a medical device in a rodent model of foreign body response. Protocols are presented to allow the host response to different immune instructive materials to be compared. The first demonstration of this capability illustrates the great potential of direct cell and tissue section analysis for in situ metabolite profiling to probe functional phenotypes using molecular signatures. Details of the in vitro cell approach, materials, sample preparation, and explant handling are presented, in addition to the data acquisition approaches and the data analysis pipelines required to achieve useful interpretation of these complex spectra. This method is useful for in situ characterization of both in vitro single cells and ex vivo tissue sections. This will aid the understanding of the immune response to medical implants by informing the design of immune-instructive biomaterials with positive interactions. It can also be used to investigate a broad range of other clinically relevant therapeutics and immune dysregulations. Graphical overview.

Conformational analysis and interaction of the Staphylococcus aureus transmembrane peptidase AgrB with its AgrD propeptide substrate.

Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.

Discovery of a polymer resistant to bacterial biofilm, swarming, and encrustation.

Innovative approaches to prevent catheter-associated urinary tract infections (CAUTIs) are urgently required. Here, we describe the discovery of an acrylate copolymer capable of resisting single- and multispecies bacterial biofilm formation, swarming, encrustation, and host protein deposition, which are major challenges associated with preventing CAUTIs. After screening ~400 acrylate polymers, poly(tert-butyl cyclohexyl acrylate) was selected for its biofilm- and encrustation-resistant properties. When combined with the swarming inhibitory poly(2-hydroxy-3-phenoxypropyl acrylate), the copolymer retained the bioinstructive properties of the respective homopolymers when challenged with Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Urinary tract catheterization causes the release of host proteins that are exploited by pathogens to colonize catheters. After preconditioning the copolymer with urine collected from patients before and after catheterization, reduced host fibrinogen deposition was observed, and resistance to diverse uropathogens was maintained. These data highlight the potential of the copolymer as a urinary catheter coating for preventing CAUTIs.

Optimization of diffuse Raman spectroscopy for in-vivo quantification of foreign body response in a small animal model.

Diffuse Raman spectroscopy (DRS) allows subsurface molecular analysis of optically turbid samples. Numerical modeling of light propagation was used as a method for improving the design of an DRS instrument to maximize the signal to noise ratio (SNR) while ensuring safe laser exposure parameters required for in-vivo measurements. Experimental validation of the model was performed on both phantom samples and disks implanted postmortem to mimic the typical response to foreign bodies (formation of a fibrotic capsule around an implant). A reduction of laser exposure of over 1500-fold was achieved over previous studies whilst maintaining the same Raman collection rates and reaching the safe power density of 3 mW/mm2. The validation of this approach in a subcutaneous implant in a mouse cadaver showed a further improvement of 1.5-fold SNR, with a thickness limit of detection for the fibrotic layer of 23 µm, under the same acquisition times. In the animal body, a thickness limit of detection of 16 µm was achieved. These results demonstrate the feasibility of numerical model-based optimization for DRS, and that the technique can be improved sufficiently to be used for in-vivo measurement of collagenous capsule formation as a result of the foreign body response in murine models.

Ink-jet 3D printing as a strategy for developing bespoke non-eluting biofilm resistant medical devices.

Chronic infection as a result of bacterial biofilm formation on implanted medical devices is a major global healthcare problem requiring new biocompatible, biofilm-resistant materials. Here we demonstrate how bespoke devices can be manufactured through ink-jet-based 3D printing using bacterial biofilm inhibiting formulations without the need for eluting antibiotics or coatings. Candidate monomers were formulated and their processability and reliability demonstrated. Formulations for in vivo evaluation of the 3D printed structures were selected on the basis of their in vitro bacterial biofilm inhibitory properties and lack of mammalian cell cytotoxicity. In vivo in a mouse implant infection model, Pseudomonas aeruginosa biofilm formation on poly-TCDMDA was reduced by ∼99% when compared with medical grade silicone. Whole mouse bioluminescence imaging and tissue immunohistochemistry revealed the ability of the printed device to modulate host immune responses as well as preventing biofilm formation on the device and infection of the surrounding tissues. Since 3D printing can be used to manufacture devices for both prototyping and clinical use, the versatility of ink-jet based 3D-printing to create personalised functional medical devices is demonstrated by the biofilm resistance of both a finger joint prosthetic and a prostatic stent printed in poly-TCDMDA towards P. aeruginosa and Staphylococcus aureus.

Pharmacological characterisation of GSK3335103, an oral αvß6 integrin small molecule RGD-mimetic inhibitor for the treatment of fibrotic disease.

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality. Activation of the pro-fibrotic cytokine transforming growth factor-ß (TGFß) via the alpha-V beta-6 (αvß6) integrin has been identified as playing a key role in the development of fibrosis. Therefore, a drug discovery programme to identify an orally bioavailable small molecule αvß6 arginyl-glycinyl-aspartic acid (RGD)-mimetic was initiated. As part of a medicinal chemistry programme GSK3335103 was identified and profiled in a range of pre-clinical in vitro and in vivo systems. GSK3335103 was shown to bind to the αvß6 with high affinity and demonstrated fast binding kinetics. In primary human lung epithelial cells, GSK3335103-induced concentration- and time-dependent internalisation of αvß6 with a rapid return of integrin to the cell surface observed after washout. Following sustained engagement of the αvß6 integrin in vitro, lysosomal degradation was induced by GSK3335103. GSK3335103 was shown to engage with the αvß6 integrin and inhibit the activation of TGFß in both ex vivo IPF tissue and in a murine model of bleomycin-induced lung fibrosis, as measured by αvß6 engagement, TGFß signalling and collagen deposition, with a prolonged duration of action observed in vivo. In summary, GSK3335103 is a potent αvß6 inhibitor that attenuates TGFß signalling in vitro and in vivo with a well-defined pharmacokinetic/pharmacodynamic relationship. This translates to a significant reduction of collagen deposition in vivo and therefore GSK3335103 represents a potential novel oral therapy for fibrotic disorders.

Chemosensitization of Temozolomide-Resistant Pediatric Diffuse Midline Glioma Using Potent Nanoencapsulated Forms of a N(3)-Propargyl Analogue.

The lack of clinical response to the alkylating agent temozolomide (TMZ) in pediatric diffuse midline/intrinsic pontine glioma (DIPG) has been associated with O6-methylguanine-DNA-methyltransferase (MGMT) expression and mismatch repair deficiency. Hence, a potent N(3)-propargyl analogue (N3P) was derived, which not only evades MGMT but also remains effective in mismatch repair deficient cells. Due to the poor pharmacokinetic profile of N3P (t1/2 < 1 h) and to bypass the blood-brain barrier, we proposed convection enhanced delivery (CED) as a method of administration to decrease dose and systemic toxicity. Moreover, to enhance N3P solubility, stability, and sustained distribution in vivo, either it was incorporated into an apoferritin (AFt) nanocage or its sulfobutyl ether ß-cyclodextrin complex was loaded into nanoliposomes (Lip). The resultant AFt-N3P and Lip-N3P nanoparticles (NPs) had hydrodynamic diameters of 14 vs 93 nm, icosahedral vs spherical morphology, negative surface charge (-17 vs -34 mV), and encapsulating ∼630 vs ∼21000 N3P molecules per NP, respectively. Both NPs showed a sustained release profile and instant uptake within 1 h incubation in vitro. In comparison to the naked drug, N3P NPs demonstrated stronger anticancer efficacy against 2D TMZ-resistant DIPG cell cultures [IC50 = 14.6 (Lip-N3P) vs 32.8 µM (N3P); DIPG-IV) and (IC50 = 101.8 (AFt-N3P) vs 111.9 µM (N3P); DIPG-VI)]. Likewise, both N3P-NPs significantly (P < 0.01) inhibited 3D spheroid growth compared to the native N3P in MGMT+ DIPG-VI (100 µM) and mismatch repair deficient DIPG-XIX (50 µM) cultures. Interestingly, the potency of TMZ was remarkably enhanced when encapsulated in AFt NPs against DIPG-IV, -VI, and -XIX spheroid cultures. Dynamic PET scans of CED-administered zirconium-89 (89Zr)-labeled AFt-NPs in rats also demonstrated substantial enhancement over free 89Zr radionuclide in terms of localized distribution kinetics and retention within the brain parenchyma. Overall, both NP formulations of N3P represent promising approaches for treatment of TMZ-resistant DIPG and merit the next phase of preclinical evaluation.

Colonisation Factor CD0873, an Attractive Oral Vaccine Candidate against Clostridioides difficile.

Clostridioides difficile is the main cause of health-care-associated infectious diarrhoea. Toxins, TcdA and TcdB, secreted by this bacterium damage colonic epithelial cells and in severe cases this culminates in pseudomembranous colitis, toxic megacolon and death. Vaccines in human trials have focused exclusively on the parenteral administration of toxin-based formulations. These vaccines promote toxin-neutralising serum antibodies but fail to confer protection from infection in the gut. An effective route to immunise against gut pathogens and stimulate a protective mucosal antibody response (secretory immunoglobulin A, IgA) at the infection site is the oral route. Additionally, oral immunisation generates systemic antibodies (IgG). Using this route, two different antigens were tested in the hamster model: The colonisation factor CD0873 and a TcdB fragment. Animals immunised with CD0873 generated a significantly higher titre of sIgA in intestinal fluid and IgG in serum compared to naive animals, which significantly inhibited the adherence of C. difficile to Caco-2 cells. Following challenge with a hypervirulent isolate, the CD0873-immunised group showed a mean increase of 80% in time to experimental endpoint compared to naïve animals. Survival and body condition correlated with bacterial clearance and reduced pathology in the cecum. Our findings advocate CD0873 as a promising oral vaccine candidate against C. difficile.

Discovery of hemocompatible bacterial biofilm-resistant copolymers.

Blood-contacting medical devices play an important role within healthcare and are required to be biocompatible, hemocompatible and resistant to microbial colonization. Here we describe a high throughput screen for copolymers with these specific properties. A series of weakly amphiphilic monomers are combinatorially polymerized with acrylate glycol monomers of varying chain lengths to create a library of 645 multi-functional candidate materials containing multiple chemical moieties that impart anti-biofilm, hemo- and immuno-compatible properties. These materials are screened in over 15,000 individual biological assays, targeting two bacterial species, one Gram negative (Pseudomonas aeruginosa) and one Gram positive (Staphylococcus aureus) commonly associated with central venous catheter infections, using 5 different measures of hemocompatibility and 6 measures of immunocompatibililty. Selected copolymers reduce platelet activation, platelet loss and leukocyte activation compared with the standard comparator PTFE as well as reducing bacterial biofilm formation in vitro by more than 82% compared with silicone. Poly(isobornyl acrylate-co-triethylene glycol methacrylate) (75:25) is identified as the optimal material across all these measures reducing P. aeruginosa biofilm formation by up to 86% in vivo in a murine foreign body infection model compared with uncoated silicone.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Gaussia Luciferase as a Reporter for Quorum Sensing in Staphylococcus aureus.

Gaussia luciferase (GLuc) is a secreted protein with significant potential for use as a reporter of gene expression in bacterial pathogenicity studies. To date there are relatively few examples of its use in bacteriology. In this study we show that GLuc can be functionally expressed in the human pathogen Staphylococcus aureus and furthermore show that it can be used as a biosensor for the agr quorum sensing (QS) system which employs autoinducing peptides to control virulence. GLuc was linked to the P3 promoter of the S. aureusagr operon. Biosensor strains were validated by evaluation of chemical agent-mediated activation and inhibition of agr. Use of GLuc enabled quantitative assessment of agr activity. This demonstrates the utility of Gaussia luciferase for in vitro monitoring of agr activation and inhibition.

Immune Modulation by Design: Using Topography to Control Human Monocyte Attachment and Macrophage Differentiation.

Macrophages play a central role in orchestrating immune responses to foreign materials, which are often responsible for the failure of implanted medical devices. Material topography is known to influence macrophage attachment and phenotype, providing opportunities for the rational design of "immune-instructive" topographies to modulate macrophage function and thus foreign body responses to biomaterials. However, no generalizable understanding of the inter-relationship between topography and cell response exists. A high throughput screening approach is therefore utilized to investigate the relationship between topography and human monocyte-derived macrophage attachment and phenotype, using a diverse library of 2176 micropatterns generated by an algorithm. This reveals that micropillars 5-10 µm in diameter play a dominant role in driving macrophage attachment compared to the many other topographies screened, an observation that aligns with studies of the interaction of macrophages with particles. Combining the pillar size with the micropillar density is found to be key in modulation of cell phenotype from pro to anti-inflammatory states. Machine learning is used to successfully build a model that correlates cell attachment and phenotype with a selection of descriptors, illustrating that materials can potentially be designed to modulate inflammatory responses for future applications in the fight against foreign body rejection of medical devices.

Whole-body and adipose tissue-specific mechanisms underlying the metabolic effects of fibroblast growth factor 21 in the Siberian hamster.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) has been shown to rapidly lower body weight in the Siberian hamster, a preclinical model of adiposity. This induced negative energy balance mediated by FGF21 is associated with both lowered caloric intake and increased energy expenditure. Previous research demonstrated that adipose tissue (AT) is one of the primary sites of FGF21 action and may be responsible for its ability to increase the whole-body metabolic rate. The present study sought to determine the relative importance of white (subcutaneous AT [sWAT] and visceral AT [vWAT]), and brown (interscapular brown AT [iBAT]) in governing FGF21-mediated metabolic improvements using the tissue-specific uptake of glucose and lipids as a proxy for metabolic activity. METHODS: We used positron emission tomography-computed tomography (PET-CT) imaging in combination with both glucose (18F-fluorodeoxyglucose) and lipid (18F-4-thiapalmitate) tracers to assess the effect of FGF21 on the tissue-specific uptake of these metabolites and compared responses to a control group pair-fed to match the food intake of the FGF21-treated group. In vivo imaging was combined with ex vivo tissue-specific functional, biochemical, and molecular analyses of the nutrient uptake and signaling pathways. RESULTS: Consistent with previous findings, FGF21 reduced body weight via reduced caloric intake and increased energy expenditure in the Siberian hamster. PET-CT studies demonstrated that FGF21 increased the uptake of glucose in BAT and WAT independently of reduced food intake and body weight as demonstrated by imaging of the pair-fed group. Furthermore, FGF21 increased glucose uptake in the primary adipocytes, confirming that these in vivo effects may be due to a direct action of FGF21 at the level of the adipocytes. Mechanistically, the effects of FGF21 are associated with activation of the ERK signaling pathway and upregulation of GLUT4 protein content in all fat depots. In response to treatment with FGF21, we observed an increase in the markers of lipolysis and lipogenesis in both the subcutaneous and visceral WAT depots. In contrast, FGF21 was only able to directly increase the uptake of lipid into BAT. CONCLUSIONS: These data identify brown and white fat depots as primary peripheral sites of action of FGF21 in promoting glucose uptake and also indicate that FGF21 selectively stimulates lipid uptake in brown fat, which may fuel thermogenesis.

Effect of adeno-associated virus (AAV)-mediated overexpression of PEPCK-M (Pck2) on Clenbuterol-induced muscle growth.

We previously identified PEPCK-M (encoded by the Pck2 gene) to be highly up-regulated in skeletal muscle of pigs treated with Ractopamine, an anabolic beta-adrenergic receptor agonist. To determine whether PEPCK-M had a causative role in modulating the skeletal muscle growth response to Ractopamine, we used adeno-associated virus 1 (AAV1) to over-express Pck2 (AAV-Pck2) in murine skeletal muscle. A contralateral limb design was employed, such that each mouse served as its own control (injected with a GFP-only expressing AAV1, labelled AAV-GFP). Daily injections of Clenbuterol (1 mg/kg for 21 days) or vehicle control were also carried out to assess the effects of AAV-Pck2 overexpression on the anabolic response to a beta-adrenergic agonist. AAV-Pck2 overexpression in leg muscles of male C57BL6/J mice for 4 weeks (6-10 weeks of age) increased Pck2 mRNA (~100-fold), protein (not quantifiable) and enzyme activity (~3-fold). There was a trend (p = 0.0798) for AAV-Pck2 overexpression to reduce TA muscle weights, but there was no significant effect on muscle fibre diameters or myosin heavy chain isoform (MyHC) mRNA expression. When skeletal muscle growth was induced by daily administration of Clenbuterol (for 21 days), overexpression of AAV-Pck2 had no effect on the growth response, nor did it alter the expression of Phosphoserine Aminotransferase-1 (Psat1) or Asparagine Synthetase (Asns) mRNA or the Clenbuterol-induced decreases in MyHC IIa and IIx mRNA expression (p = 0.0065 and p = 0.0267 respectively). However AAV-Pck2 overexpression reduced TA muscle weights (p = 0.0434), particularly in the Control (vehicle treated) mice (p = 0.059 for AAV x Clenbuterol interaction) and increased the expression of Seryl-tRNA Synthetase (Sars) mRNA (p = 0.0477). Hence, contrary to the original hypothesis, AAV-Pck2 overexpression reduced TA muscle weights and did not mimic or alter the muscle hypertrophic effects of the beta-adrenergic agonist, Clenbuterol.

Magnetically Assisted Control of Stem Cells Applied in 2D, 3D and In Situ Models of Cell Migration.

The success of cell therapy approaches is greatly dependent on the ability to precisely deliver and monitor transplanted stem cell grafts at treated sites. Iron oxide particles, traditionally used in vivo for magnetic resonance imaging (MRI), have been shown to also represent a safe and efficient in vitro labelling agent for mesenchymal stem cells (MSCs). Here, stem cells were labelled with magnetic particles, and their resulting response to magnetic forces was studied using 2D and 3D models. Labelled cells exhibited magnetic responsiveness, which promoted localised retention and patterned cell seeding when exposed to magnet arrangements in vitro. Directed migration was observed in 2D culture when adherent cells were exposed to a magnetic field, and also when cells were seeded into a 3D gel. Finally, a model of cell injection into the rodent leg was used to test the enhanced localised retention of labelled stem cells when applying magnetic forces, using whole body imaging to confirm the potential use of magnetic particles in strategies seeking to better control cell distribution for in vivo cell delivery.

Corrigendum: Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas Aeruginosa With Bronchial Epithelial Cells.

[This corrects the article DOI: 10.3389/fmicb.2018.03018.].

Contribution of the Alkylquinolone Quorum-Sensing System to the Interaction of Pseudomonas aeruginosa With Bronchial Epithelial Cells.

Pseudomonas aeruginosa causes infections in patients with compromised epithelial barrier function. Multiple virulence factors produced by P. aeruginosa are controlled by quorum sensing (QS) via 2-alkyl-4(1H)-quinolone (AQ) signal molecules. Here, we investigated the impact of AQs on P. aeruginosa PAO1 infection of differentiated human bronchial epithelial cells (HBECs). The pqsA-E operon is responsible for the biosynthesis of AQs including the 2-alkyl-3-hydroxy-4-quinolones, 4-hydroxy-2-alkylquinolines, and 4-hydroxy-2-alkylquinoline N-oxides as exemplified by pseudomonas quinolone signal (PQS), 2-heptyl-4-hydroxyquinoline (HHQ), and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), respectively. PQS and HHQ both act as QS signal molecules while HQNO is a cytochrome inhibitor. PqsE contributes both to AQ biosynthesis and promotes virulence in a PQS-independent manner. Our results show that PQS, HHQ, and HQNO were produced during PAO1 infection of HBECs, but no differences in growth or cytotoxicity were apparent when PAO1 and an AQ-negative ΔpqsA mutant were compared. Both strains promoted synthesis of inflammatory cytokines TNF-α, interleukin (IL)-6, and IL-17C by HBECs, and the provision of exogenous PQS negatively impacted on this response without affecting bacterial growth. Expression of pqsE and the PQS-independent PqsE-regulated genes mexG and lecA was detected during HBEC infection. Levels were reduced in the ΔpqsA mutant, that is, in the absence of PQS, and increased by exogenous PQS. These results support an AQ-independent role for PqsE during initial infection of HBEC by P. aeruginosa and for PQS as an enhancer of PqsE and PqsE-controlled virulence determinants and as an immunomodulator.

Antibody-Mediated Targeting of the FGFR1c Isoform Increases Glucose Uptake in White and Brown Adipose Tissue in Male Mice.

The increased prevalence of obesity and its cardiometabolic implications demonstrates the imperative to identify novel therapeutic targets able to effect meaningful metabolic changes in this population. Antibody-mediated targeting of fibroblast growth factor receptor 1c isoform (FGFR1c) has been shown to ameliorate hyperglycemia and protect from diet- and genetically-induced obesity in rodents and nonhuman primates. However, it is currently unknown which tissue(s) contribute to this glucose-lowering effect. Thus, to elucidate this effect, we treated euglycemic mice with H7, a monoclonal antibody that selectively targets FGFR1c, and used whole-body positron emission computed tomography with a glucose tracer (18F-fluorodeoxyglucose). Treatment with H7 increased basal glucose uptake in white adipose tissue (WAT), brown adipose tissue (BAT), the brain, and liver but reduced it in the quadriceps muscles. Consequentially, blood glucose was significantly reduced in response to treatment. Under insulin-stimulated conditions, the effects of H7 were maintained in WAT, BAT, liver, and muscle. Treatment with H7 decreased triglyceride (TG) content and increased adipose TG lipase content in white adipose tissue, while increasing activation of acetyl coenzyme A carboxylase, suggesting futile cycling of TGs, albeit favoring net hydrolysis. We demonstrated, in vitro, this is a direct effect of treatment in adipose tissue, as basal cellular respiration and glucose uptake were increased in response to treatment. Taken together, these data suggest that antibody-mediated targeting of FGFR1c exerts its powerful glucose-lowering efficacy primarily due to increased glucose uptake in adipose tissue.

Reduced adiposity attenuates FGF21 mediated metabolic improvements in the Siberian hamster.

FGF21 exerts profound metabolic effects in Siberian hamsters exposed to long day (LD) photoperiods that increase appetite and adiposity, however these effects are attenuated in short day (SD) animals that display hypophagia and reduced adiposity. The aim of this study was to investigate whether the beneficial effects of a novel mimetic of FGF21 in the LD state are a consequence of increased adiposity or of the central photoperiodic state. This was achieved by investigating effects of FGF21 in aged hamsters, which is associated with reduced adiposity. In LD hamsters with increased adiposity, FGF21 lowered body weight as a result of both reduced daily food intake and increased caloric expenditure, driven by an increase in whole-body fat oxidation. However, in LD animals with reduced adiposity, the effect of FGF21 on body weight, caloric intake and fat oxidation were significantly attenuated or absent when compared to those with increased adiposity. These attenuated/absent effects were underpinned by the inability of FGF21 to increase the expression of key thermogenic genes in interscapular and visceral WAT. Our study demonstrates the efficacy of a novel FGF21 mimetic in hamsters, but reveals attenuated effects in the animal model where adiposity is reduced naturally independent of photoperiod.