DOCSLIB.ORG

Properties of the C5a Receptor on Human Macrophages Vernon Seow Bachelor of Science (Honours Class 1, Microbiology)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Properties of the C5a Receptor on Human Macrophages Vernon Seow Bachelor of Science (Honours Class 1, Microbiology) Properties of the C5a Receptor on Human Macrophages Vernon Seow Bachelor of Science (Honours Class 1, Microbiology) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Institute for Molecular Bioscience I Abstract Macrophages (MΦ) are important to host defence and inflammation but plasticity in their gene expression profiles contributes to their functional heterogeneity. C5a is one of the most potent proinflammatory agents generated upon complement activation and binds to a specific receptor C5aR. Overproduction of C5a can contribute to numerous immune and inflammatory conditions. The objective of this thesis was to focus on human macrophage populations differentiated from primary human monocytes, and examine effects of C5a, C5aR agonists and antagonists, and TLR4- C5aR crosstalk on inflammatory profiles of human monocytes and macrophages. Chapter One summarizes roles for C5a activation on human macrophages, briefly surveying C5a, C5aR and some important analogues of C5a that had been developed to study roles for C5a in vivo. This chapter discusses new insights into C5aR-related biological functions and diseases, macrophage polarization and the usefulness of mouse models for human biology in relation to how closely human and mouse macrophages reproduce functional responses. Chapter Two addresses macrophage polarization. Macrophage heterogeneity was previously achieved by differentiating monocytes with either GM-CSF or M-CSF (generating GM- MΦ or M-MΦ), but was mostly studied on murine rather than human cells. This chapter describes a comparison of gene expression in populations of human macrophages (GM-MΦ versus M-MΦ), both in the basal state as well as in response to stimulation by LPS, using a combination of real-time PCR and cDNA microarray analysis, cellular migration, and functional responses. About 1000 genes are differently regulated between unstimulated GM-MΦ versus M-MΦ. Although evidence is presented in this chapter that human GM-MΦ and M-MΦ have distinct pro- and anti-inflammatory responses in human monocyte-derived macrophages, their activation by LPS supported more of a continuum without clear functional distinctions between each population. Chapter Three investigates the effects of C5aR activation on GM-MΦ versus M-MΦ using cDNA microarray. Previous reports only described gene expression in murine, rather than human, macrophages. Of ~40 genes substantially regulated by C5a in GM-MΦ and M-MΦ, 60% were common to both macrophage types. Furthermore, three different functional assays showed that C5a was equipotent on GM-MΦ and M-MΦ. These similarities for C5a-mediated functions could be due to GM-MΦ and M-MΦ having similar C5aR transcriptional and translational expression. A separate microarray experiment was conducted to overview the temporal gene expression profile induced by C5a on M-MΦ, enabling identification of rate-limiting genes as therapeutic targets in C5a-mediated inflammatory disorders. Chapter Four reports a comparative study of three known antagonists (3D53, W54011, JJ47) of C5a action via C5aR on M-MΦ. They were assessed for their relative advantages and II disadvantages as antagonists of C5aR. Traditionally, drug discovery has focused on optimizing ligand affinity for a specific receptor, enhancing functional potency, and improving drug-like properties for optimal pharmacokinetics and oral bioavailability. However, these optimization processes do not always improve in vivo efficacy. This chapter highlights some important considerations, often neglected in drug development – namely residence time, insurmountable or non-competitive antagonism for drug-receptor interactions. The non-competitive antagonist 3D53 showed superior antagonist activity compared to competitive antagonists W54011 and JJ47 despite inferior oral bioavailability, and this was because 3D53 remained bound to C5aR on macrophages for over 16h whereas compounds W54011 and JJ47 were no longer bound to C5aR after 2h. As a result, 3D53 retained high potency (IC50 20nM) in the face of competition from increasing concentrations of C5a (0.1-300nM), whereas W54011 and JJ47 were ineffective antagonists against concentrations of C5a above 30nM. The benefits of longer residence time were also demonstrated in vivo in rats. Orally administered 3D53 was an effective anti-inflammatory agent for 16h, compared to less than 2h for W54011 and JJ47, in inhibiting C5aR-mediated paw oedema in male Wistar rats. Chapter Five reports crosstalk between TLR4 and C5aR in human macrophages. C5aR is shown to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. While C5a enhanced secretion of LPS-induced IL6 and TNF from human monocytes, C5a inhibited these responses in GM-MΦ and M-MΦ. LPS amplified C5a- induced Gαi/c-Raf/MEK/ERK signalling in macrophages but not in monocytes. This synergy was independent of IL10, PI3K, p38, JNK, GM-CSF and M-CSF. C5a-mediated suppression of IL6 and TNF did not compromise but instead enhanced the clearance of Salmonella Typhimurium from macrophages. These findings implicated C5aR as a regulatory switch that modulates TLR4 signalling via the Gαi/c-Raf/MEK/ERK signalling axis in human macrophages but not in human monocytes. Chapter Six summarises all key findings in chapters 2-5, which represent important new knowledge in the field of complement C5a-C5aR and their effects on primary human monocytes and macrophages. Advances made in this thesis are specifically highlighted, discussed in relation to their novelty and significance and differences from the literature, and possible future research directions that build on the findings in this thesis are also outlined in relation to the fields of immunology and pharmacology. III Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. IV Publications during candidature Peer-reviewed papers J. Lim, A. Iyer, L. Liu, J. Y. Suen, R. J. Lohman, V. Seow, M. K. Yau, L. Brown, and D. P. Fairlie. 2013. Diet-induced obesity, adipose inflammation, and metabolic dysfunction correlating with PAR2 expression are attenuated by PAR2 antagonism. FASEB J. 27: 4757-4767. D. M. Hohenhaus, K. Schaale, K. A. Le Cao, V. Seow, A. Iyer, D. P. Fairlie, and M. J. Sweet. 2013. An mRNA atlas of G protein-coupled receptor expression during primary human monocyte/macrophage differentiation and lipopolysaccharide-mediated activation identifies targetable candidate regulators of inflammation. Immunobiology 218: 1345-1353. V. Seow, J. Lim, A. Iyer, J. Y. Suen, J. K. Ariffin, D. M. Hohenhaus, M. J. Sweet, and D. P. Fairlie. 2013. Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein c5a. J. Immunol. 191: 4308- 4316. Vesey, D. A., J. Y. Suen, V. Seow, R. J. Lohman, L. Liu, G. C. Gobe, D. W. Johnson, and D. P. Fairlie. 2013. PAR2-induced inflammatory responses in human kidney tubular epithelial cells. Am. J. Physiol. Renal Physiol. 304: F737-750. Lim, J., A. Iyer, J. Y. Suen, V. Seow, R. C. Reid, L. Brown, and D. P. Fairlie. 2013. C5aR and C3aR antagonists each inhibit diet-induced obesity, metabolic dysfunction, and adipocyte and macrophage signaling. FASEB J. 27: 822-831. V Publications included in this thesis Publication citation – incorporated as Chapter 5. V. Seow, J. Lim, A. Iyer, J. Y. Suen, J. K. Ariffin, D. M. Hohenhaus, M. J. Sweet, and D. P. Fairlie. 2013. Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein c5a. J. Immunol. 191: 4308- 4316. Contributor Statement of contribution Vernon Seow (Candidate) Designed experiments (80%) Wrote the paper (80%) Drs. Junxian Lim, Abishek Iyer, Jacky Y. Suen Designed experiments (15%) Wrote and edited paper (20%) Daniel M. Hohenhaus, Juliana K. Ariffin Designed experiments (5%) Drs. David P. Fairlie and Matthew J. Sweet Reviewed paper (100%) Publication citation – incorporated as Chapter 6 Lim, J., A. Iyer, J. Y. Suen, V. Seow, R. C. Reid, L. Brown, and D. P. Fairlie. 2013. C5aR and C3aR
Load more

Recommended publications
  • Applying Screening Techniques to Two Orphan Gpcrs
    Universidade de Lisboa Faculdade de Farmácia Deorphanization of receptors: Applying screening techniques to two orphan GPCRs Ana Catarina Rufas da Silva Santos Mestrado Integrado em Ciências Farmacêuticas 2019 Universidade de Lisboa Faculdade de Farmácia Deorphanization of receptors: Applying screening techniques to two orphan GPCRs Ana Catarina Rufas da Silva Santos Monografia de Mestrado Integrado em Ciências Farmacêuticas apresentada à Universidade de Lisboa através da Faculdade de Farmácia Orientadora: Ghazl Al Hamwi, PhD Student Co-Orientadora: Professora Doutora Elsa Maria Ribeiro dos Santos Anes, Professora Associada com Agregação em Microbiologia 2019 Abstract G-Protein Coupled Receptors represent one of the largest families of cellular receptors discovered and one of the main sources of attractive drug targets. In contrast, it also has a large number of understudied or orphan receptors. Pharmacological assays such as β-Arrestin recruitment assays, are one of the possible approaches for deorphanization of receptors. In this work, I applied the assay system previously mentioned to screen compounds in two orphan receptors, GRP37 and MRGPRX3. GPR37 has been primarily associated with a form of early onset Parkinsonism due to its’ expression patterns, and physiological role as substrate to ubiquitin E3, parkin. Although extensive literature regarding this receptor is available, the identification of a universally recognized ligand has not yet been possible. Two compounds were proposed as ligands, but both were met with controversy. These receptor association with Autosomal Recessive Juvenile Parkinson positions it as a very attractive drug target, and as such its’ deorphanization is a prime objective for investigators in this area. Regarding MRGPRX3 information is much scarcer.
    [Show full text]
  • Complement Recognition Pathways in Renal Transplantation
    BRIEF REVIEW www.jasn.org Complement Recognition Pathways in Renal Transplantation Christopher L. Nauser, Conrad A. Farrar, and Steven H. Sacks Medical Research Council Centre for Transplantation, Division of Transplantation Immunology and Mucosal Biology, King’s College London, National Health Service Guy’s and St. Thomas’ Trust, London, United Kingdom ABSTRACT The complement system, consisting of soluble and cell membrane–bound compo- weigh its effect on organ injury and nents of the innate immune system, has defined roles in the pathophysiology of renal rejection with dampening the antimi- allograft rejection. Notably, the unavoidable ischemia-reperfusion injury inherent to crobial functions of the complement sys- transplantation is mediated through the terminal complement activation products tem, such as opsonisation, cell lysis, and C5a and C5b-9. Furthermore, biologically active fragments C3a and C5a, produced recruitment of neutrophils and other in- during complement activation, can modulate both antigen presentation and T cell flammatory cells.7 It is our belief that priming, ultimately leading to allograft rejection. Earlier work identified renal tubule therapeutic strategies can be designed cell synthesis of C3, rather than hepatic synthesis of C3, as the primary source of C3 to specificallytargetthekeyinitiators driving these effects. Recent efforts have focused on identifying the local triggers of of complement activation at the relevant complement activation. Collectin-11, a soluble C-type lectin expressed in renal tis- location, such as complement-binding sue, has been implicated as an important trigger of complement activation in renal anti-HLA antibodies in the vascular tissue. In particular, collectin-11 has been shown to engage L-fucose at sites of compartment or the initiator(s) of local ischemic stress, activating the lectin complement pathway and directing the innate complement activation in the extravas- immune response to the distressed renal tubule.
    [Show full text]
  • Calpain-5 Gene Expression in the Mouse Eye and Brain
    Schaefer et al. BMC Res Notes (2017) 10:602 DOI 10.1186/s13104-017-2927-8 BMC Research Notes RESEARCH NOTE Open Access Calpain‑5 gene expression in the mouse eye and brain Kellie Schaefer1, MaryAnn Mahajan1, Anuradha Gore1, Stephen H. Tsang3, Alexander G. Bassuk4 and Vinit B. Mahajan1,2* Abstract Objective: Our objective was to characterize CAPN5 gene expression in the mouse central nervous system. Mouse brain and eye sections were probed with two high-afnity RNA oligonucleotide analogs designed to bind CAPN5 RNA and one scramble, control oligonucleotide. Images were captured in brightfeld. Results: CAPN5 RNA probes were validated on mouse breast cancer tumor tissue. In the eye, CAPN5 was expressed in the ganglion cell, inner nuclear and outer nuclear layers of the retina. Signal could not be detected in the ciliary body or the iris because of the high density of melanin. In the brain, CAPN5 was expressed in the granule cell layers of the hippocampus and cerebellum. There was scattered expression in pons. The visual cortex showed faint signal. Most signal in the brain was in a punctate pattern. Keywords: CAPN5, Calpain, In situ hybridization, Retina, Brain, Gene expression Introduction pigmentosa, retinal neovascularization, and proliferative Calpain-5 (CAPN5) is a member of the calpain family of retinopathy. Which ultimately leads to blindness [20]. calcium-activated proteases that target a variety of path- Currently there is no treatment. ways to exert control over numerous processes, includ- An important question to understanding how CAPN5 ing tissue necrosis, cytoskeletal remodeling, cell-cycle leads to disease is identifying which tissues CAPN5 is control, cell migration, myofbril turnover, regulation expressed in and the levels of CAPN5 in those tissues.
    [Show full text]
  • Edinburgh Research Explorer
    Edinburgh Research Explorer International Union of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list Citation for published version: Davenport, AP, Alexander, SPH, Sharman, JL, Pawson, AJ, Benson, HE, Monaghan, AE, Liew, WC, Mpamhanga, CP, Bonner, TI, Neubig, RR, Pin, JP, Spedding, M & Harmar, AJ 2013, 'International Union of Basic and Clinical Pharmacology. LXXXVIII. G protein-coupled receptor list: recommendations for new pairings with cognate ligands', Pharmacological reviews, vol. 65, no. 3, pp. 967-86. https://doi.org/10.1124/pr.112.007179 Digital Object Identifier (DOI): 10.1124/pr.112.007179 Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Pharmacological reviews Publisher Rights Statement: U.S. Government work not protected by U.S. copyright General rights Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer content complies with UK legislation. If you believe that the public display of this file breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 02. Oct. 2021 1521-0081/65/3/967–986$25.00 http://dx.doi.org/10.1124/pr.112.007179 PHARMACOLOGICAL REVIEWS Pharmacol Rev 65:967–986, July 2013 U.S.
    [Show full text]
  • Application Note
    APPLICATION NOTE Quantification of functional C5a using iLite® C5a Assay Ready Cells For research and professional use only. Not for use in diagnostic procedures. This application note contains a suggested protocol and performance data. Each individual laboratory must set up their own method and perform relevant validations. Background Complement component 5a (C5a) is a 74 amino acid small protein fragment of complement protein 5 (C5). C5 is cleaved to C5a and C5b by C5 convertase enzymes as a result of complement activation. Factors of the coagulation and fibrinolytic pathway are also able to cleave C5 to C5a. (1) C5a is a highly potent anaphylatoxin and chemoattracting peptide, with the ability to increase blood vessel permeability, stimulate cytokine release from myeloid cells, and expression of adhesion molecules on endothelial cells. (2) Main pro-inflammatory effector functions are induced by C5a binding to a seven-transmembrane G- protein-coupled receptor, C5aR1 (CD88). Rapidly after cleavage of C5 into C5a and C5b, C5a is metabolized by carboxypeptidases which removes the C-terminal arginine and forms C5a-desArg, a less potent ligand of C5aR1. C5a can also bind to a second receptor, C5aR2 (C5L2 or GPR77), however the biological effects associated with C5a binding C5aR2 are less well understood, both anti- inflammatory and pro-inflammatory effects have been described in literature. The C5a receptors are expressed in a wide range of cells and tissues, and the effect of C5a activation is dependent on the location of the C5a receptors. (3, 4) While the complement system is an important part of the innate immune defense against pathogens, research has shown that excessive complement activation including C5a-C5aR interaction plays a central role in several autoimmune and neurodegenerative disorders as well as in acute and chronic inflammatory conditions.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • The 'C3ar Antagonist' SB290157 Is a Partial C5ar2 Agonist
    bioRxiv preprint doi: https://doi.org/10.1101/2020.08.01.232090; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. The ‘C3aR antagonist’ SB290157 is a partial C5aR2 agonist Xaria X. Li1, Vinod Kumar1, John D. Lee1, Trent M. Woodruff1* 1School of Biomedical Sciences, The University of Queensland, St Lucia, 4072 Australia. * Correspondence: Prof. Trent M. Woodruff School of Biomedical Sciences, The University of Queensland, St Lucia, 4072 Australia. Ph: +61 7 3365 2924; Fax: +61 7 3365 1766; E-mail: [email protected] Keywords: Complement C3a, C3aR, SB290157, C5aR1, C5aR2 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.01.232090; this version posted August 3, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abbreviations used in this article: BRET, bioluminescence resonance energy transfer; BSA, bovine serum albumin; C3aR, C3a receptor C5aR1, C5a receptor 1; CHO-C3aR, Chinese hamster ovary cells stably expressing C3aR; CHO-C5aR1, Chinese hamster ovary cells stably expressing C5aR1; DMEM, Dulbecco's Modified Eagle's Medium; ERK1/2, extracellular signal-regulated kinase 1/2; FBS, foetal bovine serum; HEK293, human embryonic kidney 293 cells; HMDM, human monocyte-derived macrophage; i.p., intraperitoneal; i.v., intravenous; rhC5a, recombinant human C5a; RT, room temperature; S.E.M.
    [Show full text]
  • Primate Specific Retrotransposons, Svas, in the Evolution of Networks That Alter Brain Function
    Title: Primate specific retrotransposons, SVAs, in the evolution of networks that alter brain function. Olga Vasieva1*, Sultan Cetiner1, Abigail Savage2, Gerald G. Schumann3, Vivien J Bubb2, John P Quinn2*, 1 Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, U.K 2 Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, The University of Liverpool, Liverpool L69 3BX, UK 3 Division of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, D-63225 Germany *. Corresponding author Olga Vasieva: Institute of Integrative Biology, Department of Comparative genomics, University of Liverpool, Liverpool, L69 7ZB, [email protected] ; Tel: (+44) 151 795 4456; FAX:(+44) 151 795 4406 John Quinn: Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, The University of Liverpool, Liverpool L69 3BX, UK, [email protected]; Tel: (+44) 151 794 5498. Key words: SVA, trans-mobilisation, behaviour, brain, evolution, psychiatric disorders 1 Abstract The hominid-specific non-LTR retrotransposon termed SINE–VNTR–Alu (SVA) is the youngest of the transposable elements in the human genome. The propagation of the most ancient SVA type A took place about 13.5 Myrs ago, and the youngest SVA types appeared in the human genome after the chimpanzee divergence. Functional enrichment analysis of genes associated with SVA insertions demonstrated their strong link to multiple ontological categories attributed to brain function and the disorders. SVA types that expanded their presence in the human genome at different stages of hominoid life history were also associated with progressively evolving behavioural features that indicated a potential impact of SVA propagation on a cognitive ability of a modern human.
    [Show full text]
  • Transcriptomic Analysis of Native Versus Cultured Human and Mouse Dorsal Root Ganglia Focused on Pharmacological Targets Short
    bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. Transcriptomic analysis of native versus cultured human and mouse dorsal root ganglia focused on pharmacological targets Short title: Comparative transcriptomics of acutely dissected versus cultured DRGs Andi Wangzhou1, Lisa A. McIlvried2, Candler Paige1, Paulino Barragan-Iglesias1, Carolyn A. Guzman1, Gregory Dussor1, Pradipta R. Ray1,#, Robert W. Gereau IV2, # and Theodore J. Price1, # 1The University of Texas at Dallas, School of Behavioral and Brain Sciences and Center for Advanced Pain Studies, 800 W Campbell Rd. Richardson, TX, 75080, USA 2Washington University Pain Center and Department of Anesthesiology, Washington University School of Medicine # corresponding authors [email protected], [email protected] and [email protected] Funding: NIH grants T32DA007261 (LM); NS065926 and NS102161 (TJP); NS106953 and NS042595 (RWG). The authors declare no conflicts of interest Author Contributions Conceived of the Project: PRR, RWG IV and TJP Performed Experiments: AW, LAM, CP, PB-I Supervised Experiments: GD, RWG IV, TJP Analyzed Data: AW, LAM, CP, CAG, PRR Supervised Bioinformatics Analysis: PRR Drew Figures: AW, PRR Wrote and Edited Manuscript: AW, LAM, CP, GD, PRR, RWG IV, TJP All authors approved the final version of the manuscript. 1 bioRxiv preprint doi: https://doi.org/10.1101/766865; this version posted September 12, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • Neutrophil Chemoattractant Receptors in Health and Disease: Double-Edged Swords
    Cellular & Molecular Immunology www.nature.com/cmi REVIEW ARTICLE Neutrophil chemoattractant receptors in health and disease: double-edged swords Mieke Metzemaekers1, Mieke Gouwy1 and Paul Proost 1 Neutrophils are frontline cells of the innate immune system. These effector leukocytes are equipped with intriguing antimicrobial machinery and consequently display high cytotoxic potential. Accurate neutrophil recruitment is essential to combat microbes and to restore homeostasis, for inflammation modulation and resolution, wound healing and tissue repair. After fulfilling the appropriate effector functions, however, dampening neutrophil activation and infiltration is crucial to prevent damage to the host. In humans, chemoattractant molecules can be categorized into four biochemical families, i.e., chemotactic lipids, formyl peptides, complement anaphylatoxins and chemokines. They are critically involved in the tight regulation of neutrophil bone marrow storage and egress and in spatial and temporal neutrophil trafficking between organs. Chemoattractants function by activating dedicated heptahelical G protein-coupled receptors (GPCRs). In addition, emerging evidence suggests an important role for atypical chemoattractant receptors (ACKRs) that do not couple to G proteins in fine-tuning neutrophil migratory and functional responses. The expression levels of chemoattractant receptors are dependent on the level of neutrophil maturation and state of activation, with a pivotal modulatory role for the (inflammatory) environment. Here, we provide an overview
    [Show full text]
  • An Anticomplement Agent That Homes to the Damaged Brain and Promotes Recovery After Traumatic Brain Injury in Mice
    An anticomplement agent that homes to the damaged brain and promotes recovery after traumatic brain injury in mice Marieta M. Rusevaa,1,2, Valeria Ramagliab,1, B. Paul Morgana, and Claire L. Harrisa,3 aInstitute of Infection and Immunity, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom; and bDepartment of Genome Analysis, Academic Medical Center, Amsterdam 1105 AZ, The Netherlands Edited by Douglas T. Fearon, Cornell University, Cambridge, United Kingdom, and approved September 29, 2015 (received for review July 15, 2015) Activation of complement is a key determinant of neuropathology to rapidly and specifically inhibit MAC at sites of complement and disability after traumatic brain injury (TBI), and inhibition is activation, and test its therapeutic potential in experimental TBI. neuroprotective. However, systemic complement is essential to The construct, termed CD59-2a-CRIg, comprises CD59a linked fight infections, a critical complication of TBI. We describe a to CRIg via the murine IgG2a hinge. CD59a prevents assembly targeted complement inhibitor, comprising complement receptor of MAC in cell membranes (16), whereas CRIg binds C3b/iC3b of the Ig superfamily (CRIg) fused with complement regulator CD59a, deposited at sites of complement activation (17). The IgG2a designed to inhibit membrane attack complex (MAC) assembly at hinge promotes dimerization to increase ligand avidity. CD59- sites of C3b/iC3b deposition. CRIg and CD59a were linked via the 2a-CRIg protected in the TBI model, demonstrating that site- IgG2a hinge, yielding CD59-2a-CRIg dimer with increased iC3b/C3b targeted anti-MAC therapeutics may be effective in prevention binding avidity and MAC inhibitory activity. CD59-2a-CRIg inhibited of secondary neuropathology and improve neurologic recovery MAC formation and prevented complement-mediated lysis in vitro.
    [Show full text]
  • Retinal Pigment Epithelium Protein of 65 Kda Gene
    Molecular Vision 2013; 19:2312-2320 <http://www.molvis.org/molvis/v19/2312> © 2013 Molecular Vision Received 31 July 2013 | Accepted 14 November 2013 | Published 16 November 2013 Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/ Neuroglycan C/ chondroitin sulfate proteoglycan 5 Sandra Cottet,1,2 René Jüttner,3 Nathalie Voirol,1 Pierre Chambon,4 Fritz G. Rathjen,3 Daniel F. Schorderet,1,2,5 Pascal Escher1,2 1Institute for Research in Ophthalmology, Sion, Switzerland; 2Department of Ophthalmology, University of Lausanne, Lausanne, Switzerland; 3Max-Delbrück-Centrum, Berlin, Germany; 4Institut de Génétique et de Biologie Moléculaire et Cellulaire, Collège de France, Strasbourg, France; 5EPFL-Ecole Polytechnique Fédérale, Lausanne, Switzerland Purpose: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65−/− mouse model of Leber congenital amaurosis. Methods: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5−/−/Rpe65−/−). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Tran- scriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. Results: No differences in the progression of retinal degeneration were observed between the Rpe65−/− and Cspg5−/−/ Rpe65−/− mice.
    [Show full text]