Turning chemoattractant receptors on and off with conventional ligands and allosteric modulators: recent advances in formyl peptide receptor signaling and regulation: DOI: 10.14800/ics.73

Recruitment and activation of neutrophils at sites of infection/inflammation relies largely on the surface expression of G-protein coupled receptors (GPCRs) that recognize chemoattractants. One of these receptors, FPR1, for which formylated peptides generated by bacteria and mitochondria are high affinity agonists, was among the first human neutrophil GPCR to being cloned. This receptor shares large sequence homologies with FPR2, another member of the FPR-family expressed in human neutrophils and having a distinct ligand binding profile. The two FPRs transduce very similar neutrophil responses but possess somewhat different regulatory profiles. The FPRs have served as excellent model receptors in studies attempting to understand not only GPCR related regulation in general, but also receptor signaling in relation to innate immune reactivity and inflammation. Recent research has identified not only a large number of conventional ligands (agonist/antagonists) that regulate FPR activities by binding to surface exposed parts of the receptors, but also a number of membrane penetrating molecules that allosterically modulate receptor function after passing the membrane and interacting with the receptor on the cytosolic side. After activation, FPR signaling is rapidly terminated and the receptors become desensitized, a dormant state that can be achieved by multiple mechanisms. A coupling of the activated receptors to the actin cytoskeleton in a process that physically separates the receptors from the signaling G-protein is one such mechanism. Traditionally, the desensitized state has been viewed as a point of no return, but recent findings challenge this view and demonstrate that desensitized FPRs may in fact be reactivated to resume active signaling. The FPRs have also the capacity to communicate with other receptors in a hierarchical manner and this receptor cross-talk can both dampen and amplify neutrophil responses. In this review, we summarize some recent advances of our understanding how the FPRs can be turned on and off and discuss some future challenges, including mechanisms of allosteric modulation, receptor cross-talk, and FPR reactivation

Turning chemoattractant receptors on and off with conventional ligands and allosteric modulators: recent advances in formyl peptide receptor signaling and regulation

Recruitment and activation of neutrophils at sites of infection/inflammation relies largely on the surface expression of G-protein coupled receptors (GPCRs) that recognize chemoattractants. One of these receptors, FPR1, for which formylated peptides generated by bacteria and mitochondria are high affinity agonists, was among the first human neutrophil GPCR to being cloned. This receptor shares large sequence homologies with FPR2, another member of the FPR-family expressed in human neutrophils and having a distinct ligand binding profile. The two FPRs transduce very similar neutrophil responses but possess somewhat different regulatory profiles. The FPRs have served as excellent model receptors in studies attempting to understand not only GPCR related regulation in general, but also receptor signaling in relation to innate immune reactivity and inflammation. Recent research has identified not only a large number of conventional ligands (agonist/antagonists) that regulate FPR activities by binding to surface exposed parts of the receptors, but also a number of membrane penetrating molecules that allosterically modulate receptor function after passing the membrane and interacting with the receptor on the cytosolic side. After activation, FPR signaling is rapidly terminated and the receptors become desensitized, a dormant state that can be achieved by multiple mechanisms. A coupling of the activated receptors to the actin cytoskeleton in a process that physically separates the receptors from the signaling G-protein is one such mechanism. Traditionally, the desensitized state has been viewed as a point of no return, but recent findings challenge this view and demonstrate that desensitized FPRs may in fact be reactivated to resume active signaling. The FPRs have also the capacity to communicate with other receptors in a hierarchical manner and this receptor cross-talk can both dampen and amplify neutrophil responses. In this review, we summarize some recent advances of our understanding how the FPRs can be turned on and off and discuss some future challenges, including mechanisms of allosteric modulation, receptor cross-talk, and FPR reactivation

Porphyromonas gingivalis Produce Neutrophil Specific Chemoattractants Including Short Chain Fatty Acids

Neutrophil migration from blood to tissue-residing microbes is governed by a series of chemoattractant gradients of both endogenous and microbial origin. Periodontal disease is characterized by neutrophil accumulation in the gingival pocket, recruited by the subgingival biofilm consisting mainly of gram-negative, anaerobic and proteolytic species such as Porphyromonas gingivalis. The fact that neutrophils are the dominating cell type in the gingival pocket suggests that neutrophil-specific chemoattractants are released by subgingival bacteria, but characterization of chemoattractants released by subgingival biofilm species remains incomplete. In the present study we characterized small (< 3 kDa) soluble chemoattractants released by growing P. gingivalis, and show that these are selective for neutrophils. Most neutrophil chemoattractant receptors are expressed also by mononuclear phagocytes, the free fatty acid receptor 2 (FFAR2) being an exception. In agreement with the selective neutrophil recruitment, the chemotactic activity found in P. gingivalis supernatants was mediated in part by a mixture of short chain fatty acids (SCFAs) that are recognized by FFAR2, and other leukocytes (including monocytes) did not respond to SCFA stimulation. Although SCFAs, produced by bacterial fermentation of dietary fiber in the gut, has previously been shown to utilize FFAR2, our data demonstrate that the pronounced proteolytic metabolism employed by P. gingivalis (and likely also other subgingival biofilm bacteria associated with periodontal diseases) may result in the generation of SCFAs that attract neutrophils to the gingival pocket. This finding highlights the interaction between SCFAs and FFAR2 in the context of P. gingivalis colonization during periodontal disease, but may also have implications for other inflammatory pathologies involving proteolytic bacteria

Standardising Marine Renewable Energy Testing: Gap Analysis and Recommendations for Development of Standards

Marine renewable energy (MRE) is still an emerging technology. As such, there is still a lack of mature standards and guidance for the development and testing of these devices. The sector covers a wide range of disciplines, so there is a need for more comprehensive guidance to cover these. This paper builds on a study undertaken in the MaRINET2 project to summarise recommendations and guidance for testing MRE devices and components, by reviewing the recently published guidance. Perceived gaps in the guidance are then discussed, expanding on the previous study. Results from an industry survey are also used to help quantify and validate these gaps. The main themes identified can be summarised as: the development progression from concept to commercialisation, including more complex environmental conditions in testing, accurately modelling and quantifying the power generated, including grid integration, plus modelling and testing of novel moorings and foundation solutions. A pathway to a standardised approach to MRE testing is presented, building on recommendations learnt from the MaRINET2 round-robin testing, showing how these recommendations are being incorporated into the guidance and ultimately feeding into the development of international standards for the marine renewable energy sector

Round Robin Testing: Exploring Experimental Uncertainties through a Multifacility Comparison of a Hinged Raft Wave Energy Converter

The EU H2020 MaRINET2 project has a goal to improve the quality, robustness and accuracy of physical modelling and associated testing practices for the offshore renewable energy sector. To support this aim, a round robin scale physical modelling test programme was conducted to deploy a common wave energy converter at four wave basins operated by MaRINET2 partners. Test campaigns were conducted at each facility to a common specification and test matrix, providing the unique opportunity for intercomparison between facilities and working practices. A nonproprietary hinged raft, with a nominal scale of 1:25, was tested under a set of 12 irregular sea states. This allowed for an assessment of power output, hinge angles, mooring loads, and six-degree-of-freedom motions. The key outcome to be concluded from the results is that the facilities performed consistently, with the majority of variation linked to differences in sea state calibration. A variation of 5–10% in mean power was typical and was consistent with the variability observed in the measured significant wave heights. The tank depth (which varied from 2–5 m) showed remarkably little influence on the results, although it is noted that these tests used an aerial mooring system with the geometry unaffected by the tank depth. Similar good agreement was seen in the heave, surge, pitch and hinge angle responses. In order to maintain and improve the consistency across laboratories, we make recommendations on characterising and calibrating the tank environment and stress the importance of the device–facility physical interface (the aerial mooring in this case).</jats:p

Vertebral body stenting: a new method for vertebral augmentation versus kyphoplasty

Vertebroplasty and kyphoplasty are well-established minimally invasive treatment options for compression fractures of osteoporotic vertebral bodies. Possible procedural disadvantages, however, include incomplete fracture reduction or a significant loss of reduction after balloon tamp deflation, prior to cement injection. A new procedure called “vertebral body stenting” (VBS) was tested in vitro and compared to kyphoplasty. VBS uses a specially designed catheter-mounted stent which can be implanted and expanded inside the vertebral body. As much as 24 fresh frozen human cadaveric vertebral bodies (T11-L5) were utilized. After creating typical compression fractures, the vertebral bodies were reduced by kyphoplasty (n = 12) or by VBS (n = 12) and then stabilized with PMMA bone cement. Each step of the procedure was performed under fluoroscopic control and analysed quantitatively. Finally, static and dynamic biomechanical tests were performed. A complete initial reduction of the fractured vertebral body height was achieved by both systems. There was a significant loss of reduction after balloon deflation in kyphoplasty compared to VBS, and a significant total height gain by VBS (mean ± SD in %, p < 0.05, demonstrated by: anterior height loss after deflation in relation to preoperative height [kyphoplasty: 11.7 ± 6.2; VBS: 3.7 ± 3.8], and total anterior height gain [kyphoplasty: 8.0 ± 9.4; VBS: 13.3 ± 7.6]). Biomechanical tests showed no significant stiffness and failure load differences between systems. VBS is an innovative technique which allows for the possibly complete reduction of vertebral compression fractures and helps maintain the restored height by means of a stent. The height loss after balloon deflation is significantly decreased by using VBS compared to kyphoplasty, thus offering a new promising option for vertebral augmentation

Modulation of Receptor Signaling and Functional Selectivity in Neutrophils

Neutrophils are important effector cells of the innate immune system and in the regulation of inflammation. Many of their functions, such as chemotactic migration, secretion of granule constituents and activation of the oxygen radical-producing NADPH-oxidase, are regulated by cell surface receptors. The formyl peptide receptors (FPRs), the ATP receptor (P2Y2R) and the receptor for platelet activating factor (PAFR) belong to the large family of G-protein coupled receptors (GPCRs) and, amongst other receptors, enable neutrophils to sense and respond to host- and pathogen-derived danger signals. Therefore, any regulatory imbalance in GPCR signaling can potentially contribute to the development of severe infections or autoimmune/inflammatory diseases. The work presented in this thesis is focused on basic GPCR-signaling mechanisms in human neutrophils with the aim to generate new knowledge that could be of value for future GPCR-based drug development. To answer the scientific questions raised, numerous cell-biology-based experimental methods were applied, including measurements of neutrophil intracellular calcium release, superoxide production, degranulation, cell migration and cytoskeleton-mediated receptor regulation. The functional responses triggered by GPCRs expressed by neutrophils can be modulated in various ways at the level of receptors/ligand interaction, in dependence of other GPCRs, as well as at the signaling level. Both FPR2 and P2Y2R have been shown to be able to exert functional selective signaling through distinct regulatory mechanisms. An FPR2-specific synthetic lipopeptide allosteric modulator was identified as a biased agonist that does not induce recruitment of β-arrestin or chemotactic migration and exhibits oppositional efficacies for direct FPR2 activation and receptor cross-talk-mediated signaling. Functional selectivity liked to the P2Y2R is not related to biased agonism but instead emerges from an endogenous actin cytoskeleton-dependent regulatory mechanism which selectively inhibits the signals that lead to the generation of oxygen radicals, while leaving other signaling pathways unaffected. In conclusion, this thesis adds new knowledge to the field of neutrophil receptor biology and provides novel insights into the modulation of basic GPCR signaling mechanisms with intend to contribute to strategies for future drug design and treatment of inflammatory disorders and disease

Data on human neutrophil activation induced by pepducins with amino acid sequences derived from β2AR and CXCR4

The data described here is related to the research article titled (Gabl et al., 2016) [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR) can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from β2AR (ICL3-8) and CXCR4 (ATI-2341), respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP10 and the peptidomimetic Pam-(Lys-betaNSpe)6-NH2 (both are FPR2 specific inhibitors), but not to the FPR1 specific inhibitor cyclosporine H