key: cord-1026265-ldvspikx authors: WERNERS, A. H.; BRYANT, C. E. title: Pattern recognition receptors in equine endotoxaemia and sepsis date: 2012-05-20 journal: Equine Vet J DOI: 10.1111/j.2042-3306.2012.00574.x sha: 684c41995ad0f906c653a4830a75e759af400008 doc_id: 1026265 cord_uid: ldvspikx Pattern recognition receptors (PRRs) on host cells detect pathogens to activate innate immunity which, in turn, initiates inflammatory and adaptive immune responses. Successful activation of PRRs is, therefore, critical to controlling infections and driving pathogen‐specific adaptive immunity, but overactivity of PRRs causes systemic inflammation, which is detrimental to the host. Here we review the PRR literature as it relates to horses and speculate on the role PRRs may play in sepsis and endotoxaemia. Sepsis and/or endotoxic shock commonly accompanies conditions such as neonatal bacterial sepsis, infectious or proximal enteritis, metritis, retained placenta, colitis, strangulating gastrointestinal lesions and bacterial pneumonia [1] . Sepsis is a systemic illness caused by microbial invasion, whereas endotoxaemia occurs when endotoxin, such as lipopolysaccharide (LPS) from Gram-negative bacteria, is present in the systemic circulation [2] . Sepsis presents more challenges than endotoxaemia because bacteria express many cell surface molecules or pathogen-associated molecular patterns (PAMPs), including LPS, bacterial lipoproteins, lipoteichoic acid, peptidoglycan and bacterial DNA, many of which may be present in the circulation at one time. Traditionally, Gram-negative bacteria have been associated with sepsis; however, in humans, Gram-positive bacteria may be equally important in disease pathogenesis [3] [4] [5] . In equine neonatal sepsis, both Gram-negative and Gram-positive bacterial isolates have been identified as causative agents [6] [7] [8] [9] , and Gram-positive bacteria are increasingly being isolated from neonatal and adult animals [8] [9] [10] [11] . Pathogens and PAMPs are recognised by an extensive group of pattern recognition receptors (PRRs), each detecting specific ligands ( Table 1) . Activation of PRRs by PAMPs triggers the production of pro-and anti-inflammatory mediators, as well as initiating adaptive immune responses. Pattern recognition receptors include Toll-like receptors (TLRs), lectin receptors, Retinoic acid inducible gene I-like receptors and nucleotide-binding oligomerisation domain (NOD)-like receptors (NLRs) and may reside on the cell surface, in the endoplasmic reticulum, in endosomes, lysosomes, endolysosomes or the cytosol [12] . Successful activation of PRRs is critical in order for bacterial infections to be cleared successfully by the host, but overactivation of these receptors can lead to systemic inflammation and shock-like syndromes. Antagonism of PRRs therefore represents an exciting new therapeutic target for clinical syndromes such as sepsis and endotoxaemia [13] . Toll-like receptors are the best characterised of the PRRs. The extracellular domain of all TLRs is constructed of 19-25 leucine-rich repeats that contain hydrophobic residues at distinctive intervals to form a horseshoe structure [14, 15] . The exact structure and alignment of the different components of the leucine-rich repeats determines how ligands bind. The shapes of the binding pockets vary between species, which results in differential responses to PAMPs [16] . There are at least 10 TLRs, but in this review we will focus on only the TLRs that recognise bacteria. Mycoplasma [159] Heat-labile soluble factor (GBS-F) Group B streptococcus [160] are isolated from a clinical case, it is highly likely that this PRR will be at least partly responsible for driving any inflammatory response. 53] . In mice, TLR5 mRNA expression is found in dendritic cells, but is not detected in neutrophils [49] . In the horse, both monocytes and neutrophils express TLR5 mRNA, but show differential expression of TLR5 proteins on the cell surface [52] . Toll-like receptor 5 agonists activate equine neutrophils, but not monocytes, alveolar macrophages or peritoneal macrophages despite the fact that these cell types contain TLR5 mRNA transcripts. Cytokine gene expression induced by flagellin in neutrophils was comparable with that stimulated by LPS or Pam3CSK4 (a synthetic TLR2 agonist) [52] . What role, if any, TLR5 may play in infectious diseases in the horse is unclear, but it may be important in shock, sepsis, acute respiratory diseases and gastrointestinal infection [54] . Toll-like receptor 9 (TLR9), unlike TLRs 1, 2, 4, 5 and 6, which are all present in the cell membrane, primarily resides in the endoplasmic reticulum. It recognises unmethylated CpG containing DNA motifs from both bacteria and viruses [55] . The Cytosine-phosphate-Guanine (CpG) DNA activates dendritic cells and is important in initiating adaptive immune responses [56] . Toll-like receptor 9 forms homodimers before ligand binding [57] , then undergoes multiple cleavage steps on or after ligand binding prior to signalling [58, 59] . The precise order and timing of dimerisation and cleavage/activation remain to be established. Toll-like receptor 9 shows differential expression among normal and inflamed tissues [60] [61] [62] [63] . Equine TLR9 is found in lymphocytes, polymorphonuclear cells, bronchial epithelial cells, type II cells in the equine lung [64, 65] , and the cornea, limbus and the conjunctiva of the equine eye [66] . Age has little influence on TLR9 expression in neutrophils [67] , macrophages or dentritic cells [68] . The role of TLR9 in equine disease remains to be elucidated. Plasmodium falciparum Muramyl dipeptide (MDP) structure in peptidoglycan Gram-positive and Gram-negative bacteria [165] MurNAc-L-Ala-g-G-Glu-L-Lys (M-TRILys) Gram-positive bacteria [ OspA, outer surface protein A; NapA, neutrophil activating protein A; HSV-2, Herpes Simplex Virus-2; g-D-Glu-DAP, g-D-glutamyl-meso-diaminopimelic acid; MurNAc-L-Ala-g-G-Glu-L-Lys, N-acetylmuramic acid-L-Alanine-g-Glutamyl-L-Lysine. A. H. Werners and C. E. Bryant Nucleotide-binding oligomerisation domains (NODs) form a specific family of cytosolic receptors (NLRs), which consists of over 20 structurally related proteins [69] . There are 2 distinct families of NLRs; the NLRP proteins contain a pyrin domain, and the NLRC proteins, such as NOD1, NOD2, NLRC3 and NLRC4 contain a caspase recruitment domain [70] . NLRP3, NLRP1 and NLRC4 form protein complexes called inflammasomes such that upon ligand binding a change in NLR confirmation leads to recruitment of an adaptor molecule (apoptosis-related speck-like protein [ASC]) and an effector molecule (pro-caspase-1) in an oligomeric complex. This complex activates a proteolytic cascade resulting in the maturation and release of, amongst others, proinflammatory cytokines of the interleukin-1 family [71] . The NLRs are emerging as very important therapeutic targets in many inflammatory diseases in humans. Both NOD1 and NOD2 recognise bacterial ligands [72, 73] . Whereas NOD1 is ubiquitously expressed, NOD2 is expressed only in monocytes, macrophages, dendritic cells and intestinal epithelial cells [74] . A peptidoglycan derivative (L-Ala-D-Glu-meso-DAP [diaminopimelic acid]), present in almost all Gram-negative bacteria, is recognised by NOD1 [75, 76] . However, NOD2 detects M-dipeptide and GM-dipeptide, both of which are degradation products of peptidoglycans. GM-dipeptide is found in all bacteria, hence NOD2 can be regarded as a general sensor of peptidoglycan degradation products [72] . There are limited data on equine NOD1 and NOD2, but horses with Recurrent Airway Obstruction show upregulation of NOD2-induced nuclear factor-kB activation [77] . The receptor NLRC4 (also known as IPAF) is expressed in myeloid cells [78, 79] . It recognises a variety of pathogens, including S. Typhimurium [80] , Pseudomonas aeruginosa [81] , Shigella [82] , Legionella pneomophila [83] , bacterial flagellin [84, 85] and a basal rod component of some bacterial type III secretion systems [86] . This receptor is present in the equine genome, but it is unclear what role it might play in equine bacterial diseases. Many 'danger signals' are recognised by NLRP3 (cryopyrin or NALP3), both infectious (for example recognising Staphylococcus aureus [87] , Staphylococcus pneumoniae [88] and S. Typhmuium [89] ) as well as noninfectious, endogenous or exogenous molecules. The wide variety and diverse nature of these ligands suggest it is unlikely that they interact directly with NLRP3, but trigger inflammasome formation indirectly [90] . It is important in many chronic inflammatory syndromes in humans, and it is present in the equine genome. A fourth inflammasome complex is formed by association of a pyrin and HIN200 domain containing protein family member (absent in melanoma 2 [AIM2]) with ASC and caspase-1 [90] . A cytosolic receptor, AIM2, recognises double-stranded DNA [91] [92] [93] and is an important sensor for bacterial double-stranded DNA from both Listeria monocytogenes and Francisella tularensis [94, 95] . It is present in a limited number of mammalian species, of which the horse is one, and therefore this PRR is also potentially important in horses. Pattern recognition receptor agonists (for adjuvants) and antagonists are under development. Some antagonists at other PRRs have been described, but antagonists of TLR4 and TLR2 are likely to be most useful in equine endotoxaemia and sepsis. Antagonism at TLR4 is the most obvious therapeutic target for equine endotoxaemia and sepsis. Development of TLR4 antagonists is challenging because many of the drugs developed are derived from bacterial lipid As that are antagonists in humans and mice, but this does not mean they will necessarily be antagonists in the horse. Lipopolysaccharide from Rhodobacter sphaeroides, for example, is a TLR4 antagonist in humans and mice, but it is an agonist in the horse and hamster [96, 97] . E5531, a synthetic compound based on the lipid A structure of Rhodobacter capsulatus, is an antagonist in mice and humans, an antagonist in equine cell models, but an agonist in an equine whole-blood model [98] [99] [100] . A second-generation compound based on E5531, eritoran (E5564), is a potent antagonist of LPS in humans [101, 102] and in horses [103] , but in phase III clinical trials [104] it did not meet its primary end-point in humans with severe sepsis [105] . Several other TLR4 antagonists are currently being investigated in humans and mice for the treatment of different acute and chronic inflammatory diseases [13, 106] . Antagonistic phospolipids for TLR2 have been synthesised, but currently there is little information available beyond their initial description [107] . Toll-like receptor 2 antibodies protect mice from lethal septic shock syndrome [108] , and anti-TLR2 antibodies that prevent trafficking of the receptor from the endoplasmic reticulum to the cell surface were shown to inhibit in vitro and ex vivo ligand-driven cell activation [109] . Anti-TLR2 antibodies also show beneficial effects in arthritis and ischaemia-reperfusion injury models [110, 111] , but it is unlikely that commercial equine-specific TLR-blocking antibodies will be developed for horses. It is likely, however, that TLR2 antagonists may be useful for a range of equine conditions, for example, neonatal diarrhoea-associated sepsis, should these compounds become available for use in horses. In conclusion, PRRs that recognise bacteria are likely to be useful therapeutic targets for treating equine sepsis and endotoxaemia. However, PRR antagonists will need careful clinical evaluation because of the controversial results emerging from human clinical trials due to the complex, multifactorial pathogenesis of these diseases. Use of TLR4 antagonists in endotoxic horses is likely to be successful, but whether PRR antagonists will be useful in septic foals is less clear. Infections with mixed bacterial species will potentially involve multiple PRRs, suggesting that combination therapy simultaneously inhibiting several PRRs may be necessary. Complete inhibition of PRRs is potentially detrimental, particularly in sepsis, because TLR4 and TLR2 knockout mice show increased mortality in response to Gram-negative or Gram-positive bacteria, respectively. Specific equine drugs will need to be developed to achieve a safe treatment that blocks systemic inflammation whilst retaining the protective immune responses against bacterial infection. No conflicts of interest have been declared. 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asbestos and silica Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome The NALP3 inflammasome is involved in the innate immune response to amyloid-b A novel role for the NLRC4 inflammasome in mucosal defenses against the fungal pathogen Candida albicans Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages The authors would like to thank the Horserace Betting Levy Board (HBLB) for their support of CEBs research. Stay up-to-date on the latest advances and current issues in equine medicine with this handy reference for the busy equine practitioner, large animal veterinarian or student. This edition of Current Therapy in Equine Medicine brings you thorough coverage and expert advice on selected topics in areas that have seen significant advances in the last 5 years. Content emphasises the practical aspects of diagnosis and treatment and provides details for therapeutic regimens. Arranged primarily by body system, the text also features sections on infectious diseases, foal diseases, nutrition and toxicology. With this cutting-edge information all in one reliable source, you'll increase your awareness of key therapies in less time. Publisher: Saunders, January 2010 • Hardback, 1488 pages Develop an essential understanding of the principles of equine disease with this one-of-a-kind, problem-based resource! Extensively revised and updated with contributions from an international team of experts, this 3rd edition reflects the latest clinical research in equine medicine and focuses on the basic pathophysiological mechanisms that underlie the development of various equine diseases to help you confidently diagnose, treat and manage patient conditions. Includes a bound-in companion DVD containing more than 120 high-quality video clips that guide you through procedures related to the cardiovascular and neurological systems.