Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo.

Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic.

Galectin-3: A Positive Regulator of Leukocyte Recruitment in the Inflamed Microcirculation.

In vivo and ex vivo imaging were used to investigate the function of galectin-3 (Gal-3) during the process of leukocyte recruitment to the inflamed microcirculation. The cremasteric microcirculation of wild-type (C57BL/6), Gal-3-/-, and CX3CR1gfp/+ mice were assessed by intravital microscopy after PBS, IL-1ß, TNF-α, or recombinant Gal-3 treatment. These cellular responses were investigated further using flow-chamber assays, confocal microscopy, flow cytometry, PCR analysis, and proteome array. We show that mechanisms mediating leukocyte slow rolling and emigration are impaired in Gal-3-/- mice, which could be because of impaired expression of cell adhesion molecules and an altered cell surface glycoproteome. Local (intrascrotal) administration of recombinant Gal-3 to wild-type mice resulted in a dose-dependent reduction in rolling velocity associated with increased numbers of adherent and emigrated leukocytes, ∼50% of which were Ly6G+ neutrophils. Intrascrotal administration of Gal-3 to CX3CR1gfp/+ mice confirmed that approximately equal numbers of monocytes are also recruited in response to this lectin. Exogenous Gal-3 treatment was accompanied by increased proinflammatory cytokines and chemokines within the local tissue. In conclusion, this study unveils novel biology for both exogenous and endogenous Gal-3 in promoting leukocyte recruitment during acute inflammation.

Endothelial Cell Junctional Adhesion Molecules: Role and Regulation of Expression in Inflammation.

Endothelial cells line the lumen of all blood vessels and play a critical role in maintaining the barrier function of the vasculature. Sealing of the vessel wall between adjacent endothelial cells is facilitated by interactions involving junctionally expressed transmembrane proteins, including tight junctional molecules, such as members of the junctional adhesion molecule family, components of adherence junctions, such as VE-Cadherin, and other molecules, such as platelet endothelial cell adhesion molecule. Of importance, a growing body of evidence indicates that the expression of these molecules is regulated in a spatiotemporal manner during inflammation: responses that have significant implications for the barrier function of blood vessels against blood-borne macromolecules and transmigrating leukocytes. This review summarizes key aspects of our current understanding of the dynamics and mechanisms that regulate the expression of endothelial cells junctional molecules during inflammation and discusses the associated functional implications of such events in acute and chronic scenarios.

Superoxide generation and leukocyte accumulation: key elements in the mediation of leukotriene B4-induced itch by transient receptor potential ankyrin 1 and transient receptor potential vanilloid 1.

The underlying mechanisms of itch are poorly understood. We have investigated a model involving the chemoattractant leukotriene B4 (LTB4) that is up-regulated in common skin diseases. Intradermal injection of LTB4 (0.1 nmol/site) into female CD1 mice induced significant scratching movements (used as an itch index) compared with vehicle-injected (0.1% bovine serum albumin-saline) mice. Intraperitoneal transient receptor potential (TRP) channel antagonist treatment significantly inhibited itch as follows: TRP vanilloid 1 (TRPV1) antagonist SB366791 (0.5 mg/kg, by 97%) and the TRP ankyrin 1 (TRPA1) antagonists TCS 5861528 (10 mg/kg; 82%) and HC-030031 (100 mg/kg; 76%). Leukotriene B4 receptor 2 antagonism by LY255283 (5 mg/kg i.p.; 62%) reduced itch. Neither TRPV1-knockout (TRPV1-KO) nor TRPA1-knockout (TRPA1-KO mice exhibited LTB4-induced itch compared with their wild-type counterparts. The reactive oxygen species scavengers N-acetylcysteine (NAC; 204 mg/kg i.p.; 86%) or superoxide dismutase (SOD; 10 mg/kg i.p.; 83%) also inhibited itch. LTB4-induced superoxide release was attenuated by TCS 5861528 (56%) and HC-030031 (66%), NAC (58%), SOD (50%), and LY255283 (59%) but not by the leukotriene B4 receptor 1 antagonist U-75302 (9 nmol/site) or SB366791. Itch, superoxide, and myeloperoxidase generation were inhibited by the leukocyte migration inhibitor fucoidan (10 mg/kg i.v.) by 80, 61, and 34%, respectively. Myeloperoxidase activity was also reduced by SB366791 (35%) and SOD (28%). TRPV1-KO mice showed impaired myeloperoxidase release, whereas TRPA1-KO mice exhibited diminished production of superoxide. This result provides novel evidence that TRPA1 and TRPV1 contribute to itch via distinct mechanisms.

Design and pharmacological evaluation of PF-4840154, a non-electrophilic reference agonist of the TrpA1 channel.

TrpA1 is an ion channel involved in nociceptive and inflammatory pain. It is implicated in the detection of chemical irritants through covalent binding to a cysteine-rich intracellular region of the protein. While performing an HTS of the Pfizer chemical collection, a class of pyrimidines emerged as a non-reactive, non-covalently binding family of agonists of the rat and human TrpA1 channel. Given the issues identified with the reference agonist Mustard Oil (MO) in screening, a new, non-covalently binding agonist was optimized and proved to be a superior agent to MO for screening purposes. Compound 16a (PF-4840154) is a potent, selective agonist of the rat and human TrpA1 channel and elicited TrpA1-mediated nocifensive behaviour in mouse.

Environmental cold exposure increases blood flow and affects pain sensitivity in the knee joints of CFA-induced arthritic mice in a TRPA1-dependent manner.

BACKGROUND: The effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor. METHODS: Mono-arthritis was induced by unilateral intra-articular injection of complete Freund's adjuvant (CFA) in CD1 mice, and in mice either lacking TRPA1 (TRPA1 KO) or respective wildtypes (WT). Two weeks later, nociception and joint blood flow were measured following exposure to 10 °C (1 h) or room temperature (RT). Primary mechanical hyperalgesia in the knee was measured by pressure application apparatus; secondary mechanical hyperalgesia by automated von Frey system; thermal hyperalgesia by Hargreaves technique, and weight bearing by the incapacitance test. Joint blood flow was recorded by full-field laser perfusion imager (FLPI) and using clearance of (99m)Technetium. Blood flow was assessed after pretreatment with antagonists of either TRPA1 (HC-030031), substance P neurokinin 1 (NK1) receptors (SR140333) or calcitonin gene-related peptide (CGRP) (CGRP8-37). TRPA1, TAC-1 and CGRP mRNA levels were examined in dorsal root ganglia, synovial membrane and patellar cartilage samples. RESULTS: Cold exposure caused bilateral primary mechanical hyperalgesia 2 weeks after CFA injection, in a TRPA1-dependent manner. In animals maintained at RT, clearance techniques and FLPI showed that CFA-treated joints exhibited lower blood flow than saline-treated joints. In cold-exposed animals, this reduction in blood flow disappears, and increased blood flow in the CFA-treated joint is observed using FLPI. Cold-induced increased blood flow in CFA-treated joints was blocked by HC-030031 and not observed in TRPA1 KOs. Cold exposure increased TRPA1 mRNA levels in patellar cartilage, whilst reducing it in synovial membranes from CFA-treated joints. CONCLUSIONS: We provide evidence that environmental cold exposure enhances pain and increases blood flow in a mono-arthritis model. These changes are dependent on TRPA1. Thus, TRPA1 may act locally within the joint to influence blood flow via sensory nerves, in addition to its established nociceptive actions.

Investigating the potential role of TRPA1 in locomotion and cardiovascular control during hypertension.

Radiotelemetry was used to investigate the in vivo cardiovascular and activity phenotype of both TRPA1 (transient receptor potential ankyrin 1) wild-type (WT) and TRPA1 knockout (KO) mice. After baseline recording, experimental hypertension was induced using angiotensin II infusion (1.1 mg(-1) kg(-1) a day, for 14 days). TRPA1 WT and KO mice showed similar morphological and functional cardiovascular parameters, including similar basal blood pressure (BP), heart rate, size, and function. Similar hypertension was also displayed in response to angiotensin II (156 ± 7 and 165 ± 11 mmHg, systolic BP ± SEM, n = 5-6). TRPA1 KO mice showed increased hypertensive hypertrophy (heart weight:tibia length: 7.3 ± 1.6 mg mm(-1) vs. 8.8 ± 1.7 mg mm(-1)) and presented with blunted interleukin 6 (IL-6) production compared with hypertensive WT mice (151 ± 24 vs. 89 ± 16 pg mL(-1)). TRPA1 expression in dorsal root ganglion (DRG) neurones was upregulated during hypertension (163% of baseline expression). Investigations utilizing the TRPA1 agonist cinnamaldehyde (CA) on mesenteric arterioles isolated from näive mice suggested a lack of TRPA1-dependent vasoreactivity in this vascular bed; a site with notable ability to alter total peripheral resistance. However, mesenteric arterioles isolated from TRPA1 KO hypertensive mice displayed significantly reduced ability to relax in response to nitric oxide (NO) (P < 0.05). Unexpectedly, naïve TRPA1 KO mice also displayed physical hyperactivity traits at baseline, which was exacerbated during hypertension. In conclusion, our study provides a novel cardiovascular characterization of TRPA1 KO mice in a model of hypertension. Results suggest that TRPA1 has a limited role in global cardiovascular control, but we demonstrate an unexpected capacity for TRPA1 to regulate physical activity.

TRPA1 is essential for the vascular response to environmental cold exposure.

The cold-induced vascular response, consisting of vasoconstriction followed by vasodilatation, is critical for protecting the cutaneous tissues against cold injury. Whilst this physiological reflex response is historic knowledge, the mechanisms involved are unclear. Here by using a murine model of local environmental cold exposure, we show that TRPA1 acts as a primary vascular cold sensor, as determined through TRPA1 pharmacological antagonism or gene deletion. The initial cold-induced vasoconstriction is mediated via TRPA1-dependent superoxide production that stimulates α2C-adrenoceptors and Rho-kinase-mediated MLC phosphorylation, downstream of TRPA1 activation. The subsequent restorative blood flow component is also dependent on TRPA1 activation being mediated by sensory nerve-derived dilator neuropeptides CGRP and substance P, and also nNOS-derived NO. The results allow a new understanding of the importance of TRPA1 in cold exposure and provide impetus for further research into developing therapeutic agents aimed at the local protection of the skin in disease and adverse climates.

TRPV1 and SP: key elements for sepsis outcome?

UNLABELLED: Sensory neurons play important roles in many disorders, including inflammatory diseases, such as sepsis. Sepsis is a potentially lethal systemic inflammatory reaction to a local bacterial infection, affecting thousands of patients annually. Although associated with a high mortality rate, sepsis outcome depends on the severity of systemic inflammation, which can be directly influenced by several factors, including the immune response of the patient. Currently, there is a lack of effective drugs to treat sepsis, and thus there is a need to develop new drugs to improve sepsis outcome. Several mediators involved in the formation of sepsis have now been identified, but the mechanisms underlying the pathology remain poorly understood. The transient receptor potential vanilloid 1 (TRPV1) receptor and the neuropeptide substance P (SP) have recently been demonstrated as important targets for sepsis and are located on sensory neurones and non-neuronal cells. Herein, we highlight and review the importance of sensory neurones for the modulation of sepsis, with specific focus on recent findings relating to TRPV1 and SP, with their distinct abilities to alter the transition from local to systemic inflammation and also modify the overall sepsis outcome. We also emphasize the protective role of TRPV1 in this context. LINKED ARTICLES: This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7.

A role for TRPV1 in influencing the onset of cardiovascular disease in obesity.

Obesity induced by Western diets is associated with type 2 diabetes mellitus and cardiovascular diseases, although underlying mechanisms are unclear. We investigated a murine model of diet-induced obesity to determine the effect of transient potential receptor vanilloid 1 (TRPV1) deletion on hypertension and metabolic syndrome. Wild-type and TRPV1 knockout mice were fed normal or high-fat diet from 3 to 15 weeks. High-fat diet-fed mice from both genotypes became obese, with similar increases in body and adipose tissue weights. High-fat diet-fed TRPV1 knockout mice showed significantly improved handling of glucose compared with high-fat diet-fed wild-type mice. Hypertension, vascular hypertrophy, and altered nociception were observed in high-fat diet-fed wild-type but not high-fat diet-fed TRPV1 knockout mice. Wild-type, but not high-fat diet-fed TRPV1 knockout, mice demonstrated remodeling in terms of aortic vascular hypertrophy and increased heart and kidney weight, although resistance vessel responses were similar in each. Moreover, the wild-type mice had significantly increased plasma levels of leptin, interleukin 10 and interleukin 1ß, whereas samples from TRPV1 knockout mice did not show significant increases. Our results do not support the concept that TRPV1 plays a major role in influencing weight gain. However, we identified a role of TRPV1 in the deleterious effects observed with high-fat feeding in terms of inducing hypertension, impairing thermal nociception sensitivity, and reducing glucose tolerance. The observation of raised levels of adipokines in wild-type but not TRPV1 knockout mice is in keeping with TRPV1 involvement in stimulating the proinflammatory network that is central to obesity-induced hypertension and sensory neuronal dysfunction.

Neutrophil heterogeneity in health and disease: a revitalized avenue in inflammation and immunity.

Leucocytes form the principal cellular components of immunity and inflammation, existing as multiple subsets defined by distinct phenotypic and functional profiles. To date, this has most notably been documented for lymphocytes and monocytes. In contrast, as neutrophils are traditionally considered, to be short-lived, terminally differentiated cells that do not re-circulate, the potential existence of distinct neutrophil subsets with functional and phenotypic heterogeneity has not been widely considered or explored. A growing body of evidence is now challenging this scenario, and there is significant evidence for the existence of different neutrophil subsets under both physiological and pathological conditions. This review will summarize the key findings that have triggered a renewed interest in neutrophil phenotypic changes, both in terms of functional implications and consequences within disease models. Special emphasis will be placed on the potential pro- and anti-inflammatory roles of neutrophil subsets, as indicated by the recent works in models of ischaemia-reperfusion injury, trauma, cancer and sepsis.

The vasoactive potential of kisspeptin-10 in the peripheral vasculature.

Splice products of the Kiss1 protein (kisspeptins) have been shown to be involved in a diverse range of functions, including puberty, metastasis and vasoconstriction in large human arteries. Circulating Kisspeptin-10 (Kp-10) plasma levels are low in normal individuals but are elevated during various disease states as well as pregnancy. Here, we investigated the potential of Kp-10, the shortest biologically active kisspeptin, to influence microvascular effects, concentrating on the cutaneous vasculature. Kp-10 caused a dose-dependent increase in oedema formation (0.3-10 nmol/injection site), assessed by Evans Blue albumin dye extravasation, in the dorsal skin of CD1 mice. Oedema formation was shown to be inhibited by the histamine H(1) receptor antagonist mepyramine. The response was characterised by a ring of pallor at the injection site in keeping with vasoconstrictor activity. Therefore, changes in dorsal skin blood flow were assessed by clearance of intradermally injected (99m)technetium. Kp-10 was found to significantly reduce clearance, in keeping with decreased blood flow and providing further evidence for vasoconstrictor activity. The decreased clearance was partially inhibited by co-treatment with the cyclo-oxygenase inhibitor indomethacin. Finally evidence for the kisspeptin receptor gene (Kiss1R), but not the kisspeptin peptide gene (Kiss1), mRNA expression was observed in heart, aorta and kidney samples from normal and angiotensin II induced hypertensive mice, with similar mRNA levels observed in each. We have evidence for two peripheral vasoactive roles for kisspeptin-10. Firstly, plasma extravasation indicative of ability to induce oedema formation and secondly decreased peripheral blood flow, indicating microvascular constriction. Thus Kp-10 has vasoactive properties in the peripheral microvasculature.

Evidence for the pathophysiological relevance of TRPA1 receptors in the cardiovascular system in vivo.

AIMS: The aim of the study is to investigate transient receptor potential ankyrin 1 (TRPA1)-induced responses in the vasculature and on blood pressure and heart rate (HR), in response to TRPA1 agonists using wild-type (WT) and TRPA1 knockout (KO) mice. METHODS AND RESULTS: TRPA1 agonists allyl isothiocyanate and cinnamaldehyde (CA) significantly increased blood flow in the skin of anaesthetized WT, but not in TRPA1 KO mice. CA also induced TRPA1-dependent relaxation of mesenteric arteries. Intravenously injected CA induced a transient hypotensive response accompanied by decreased HR that was, depending on genotype and dose, followed by a more sustained dose-dependent pressor response (10-320 micromol/kg). CA (80 micromol/kg) induced a depressor response that was significantly less in TRPA1 KO mice, with minimal pressor effects. The pressor response of a higher CA dose (320 micromol/kg) was observed in WT but not in TRPA1 KO mice, indicating involvement of TRPA1. Experiments using TRP vanilloid 1 (TRPV1) KO and calcitonin gene-related peptide (CGRP) KO mice provided little evidence for the involvement of TRPV1 or CGRP, nor did blocking substance P receptors affect responses. However, the cholinergic antagonist atropine sulphate (5 mg/kg) significantly inhibited the depressor response and slowed HR with CA (80 micromol/kg), but had no effect on pressor responses. The pressor response remained unaffected, even in the presence of the ganglion blocker hexamethonium bromide (1 mg/kg). The alpha-adrenergic blocker prazosin hydrochloride (1 mg/kg) significantly inhibited both components, but not slowed HR. CONCLUSION: TRPA1 is involved in mediating vasodilation. TRPA1 can also influence changes in blood pressure of possible relevance to autonomic system reflexes and potentially to vasovagal/neurocardiogenic syncope disorders.

Hydrogen peroxide is a novel mediator of inflammatory hyperalgesia, acting via transient receptor potential vanilloid 1-dependent and independent mechanisms.

Inflammatory diseases associated with pain are often difficult to treat in the clinic due to insufficient understanding of the nociceptive pathways involved. Recently, there has been considerable interest in the role of reactive oxygen species (ROS) in inflammatory disease, but little is known of the role of hydrogen peroxide (H(2)O(2)) in hyperalgesia. In the present study, intraplantar injection of H(2)O(2)-induced a significant dose- and time-dependent mechanical and thermal hyperalgesia in the mouse hind paw, with increased c-fos activity observed in the dorsal horn of the spinal cord. H(2)O(2) also induced significant nociceptive behavior such as increased paw licking and decreased body liftings. H(2)O(2) levels were significantly raised in the carrageenan-induced hind paw inflammation model, showing that this ROS is produced endogenously in a model of inflammation. Moreover, superoxide dismutase and catalase significantly reduced carrageenan-induced mechanical and thermal hyperalgesia, providing evidence of a functionally significant endogenous role. Thermal, but not mechanical, hyperalgesia in response to H(2)O(2) (i.pl.) was longer lasting in TRPV1 wild type mice compared to TRPV1 knockouts. It is unlikely that downstream lipid peroxidation was increased by H(2)O(2). In conclusion, we demonstrate a notable effect of H(2)O(2) in mediating inflammatory hyperalgesia, thus highlighting H(2)O(2) removal as a novel therapeutic target for anti-hyperalgesic drugs in the clinic.

The Sharpless asymmetric aminohydroxylation reaction: optimising ligand/substrate control of regioselectivity for the synthesis of 3- and 4-aminosugars.

An investigation of the factors responsible for the sense and magnitude of regioselectivity in the Sharpless asymmetric aminohydroxylation (AA) has been conducted. Theoretical investigations of ligand-osmium binding geometry and experimental investigations of the Sharpless AA reaction on a series of functionalized pent-2-enoic acid ester substrates demonstrate that the opposite regioselectivity afforded using PHAL and AQN ligands results from a change in substrate orientation with respect to the catalyst. Two distinct ligand binding domains within the catalyst have been proposed that undergo attractive interactions with the substrates. Selective access to each of the four potential regio- and stereo-isomeric AA products could be achieved through the appropriate choice of ligand and substrate. These results have been applied toward the efficient stereoselective synthesis of naturally occurring and regioisomeric 3- and 4-aminosugar derivatives.

Antifungal effects on metabolite profiles of medically important yeast species measured by nuclear magnetic resonance spectroscopy.

Drug-induced inhibition of fungal growth is used in the diagnostic laboratory to predict therapeutic efficacy but is relatively slow, and determination of endpoints can be problematic. Nuclear magnetic resonance (NMR) spectroscopy identifies the metabolic complement of microorganisms while monitoring utilization of constituents of the incubation medium. This technique may provide a rapid and objective indicator of antifungal effects. We evaluated the effects of caspofungin, amphotericin B (AMB), and voriconazole on metabolic profiles of yeast species cultured in RPMI-2% glucose-morpholinepropanesulfonic acid buffer in microtiter plates in a proof-of-principle study. 1H NMR spectra were obtained using Bruker NMR spectrometers at 1H frequencies of 600 and 360 MHz. Metabolites were identified by two-dimensional correlation NMR spectra, and relative peak integrals were calculated from one-dimensional 1H NMR spectra. MICs were determined by a modification of the Clinical and Laboratory Standards Institute broth microdilution method M27-A. Utilization of glucose and branched-chain and aromatic amino acid substrates was accompanied by fungal production of acetate, acetaldehyde, ethanol, formate, fumarate, glycerol, lactate, pyruvate, and succinate. Clear-cut metabolic endpoints indicating a greater than 50% reduction in substrate utilization and fungal metabolite production which correlated with MICs were noted at 16 and 24 h for all three drugs. At 8 h, reductions of greater than 50% for selected metabolites were noted for caspofungin and AMB. Direct NMR-based observation of metabolic alterations in yeast cultures reveals changes in key metabolic pathways and should be evaluated formally as a rapid technique for determining susceptibility to antifungal drugs.