CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signaling.

Introduction: CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterize this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. Methods: In the absence of structural information, in silico modeling techniques assessed the likelihood of CXCL17 adopting a chemokine fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils, and CXCR1 transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. Results: All modeling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated owing to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. Discussion: In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

Structure-activity studies of Streptococcus pyogenes enzyme SpyCEP reveal high affinity for CXCL8 in the SpyCEP C-terminal.

The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR+ CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (KM) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally-truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar KM of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2200-fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity.

The protease associated (PA) domain in ScpA from Streptococcus pyogenes plays a role in substrate recruitment.

Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage.

Virtually screening adults for depression, anxiety, and suicide risk using machine learning and language from an open-ended interview.

Background: Current depression, anxiety, and suicide screening techniques rely on retrospective patient reported symptoms to standardized scales. A qualitative approach to screening combined with the innovation of natural language processing (NLP) and machine learning (ML) methods have shown promise to enhance person-centeredness while detecting depression, anxiety, and suicide risk from in-the-moment patient language derived from an open-ended brief interview. Objective: To evaluate the performance of NLP/ML models to identify depression, anxiety, and suicide risk from a single 5-10-min semi-structured interview with a large, national sample. Method: Two thousand four hundred sixteen interviews were conducted with 1,433 participants over a teleconference platform, with 861 (35.6%), 863 (35.7%), and 838 (34.7%) sessions screening positive for depression, anxiety, and suicide risk, respectively. Participants completed an interview over a teleconference platform to collect language about the participants' feelings and emotional state. Logistic regression (LR), support vector machine (SVM), and extreme gradient boosting (XGB) models were trained for each condition using term frequency-inverse document frequency features from the participants' language. Models were primarily evaluated with the area under the receiver operating characteristic curve (AUC). Results: The best discriminative ability was found when identifying depression with an SVM model (AUC = 0.77; 95% CI = 0.75-0.79), followed by anxiety with an LR model (AUC = 0.74; 95% CI = 0.72-0.76), and an SVM for suicide risk (AUC = 0.70; 95% CI = 0.68-0.72). Model performance was generally best with more severe depression, anxiety, or suicide risk. Performance improved when individuals with lifetime but no suicide risk in the past 3 months were considered controls. Conclusion: It is feasible to use a virtual platform to simultaneously screen for depression, anxiety, and suicide risk using a 5-to-10-min interview. The NLP/ML models performed with good discrimination in the identification of depression, anxiety, and suicide risk. Although the utility of suicide risk classification in clinical settings is still undetermined and suicide risk classification had the lowest performance, the result taken together with the qualitative responses from the interview can better inform clinical decision-making by providing additional drivers associated with suicide risk.

What defines a chemokine? - The curious case of CXCL17.

Chemotactic cytokines (chemokines) are a group of around 40 small proteins which share a similar protein fold and are well known for their ability to direct the migration of leukocytes to a variety of tissue locations. CXCL17 was the last member of the chemokine family to be assigned and was admitted to the family based on theoretical modelling of the CXCL17 structure and chemotactic activity for monocytes and dendritic cells. Of Interest, CXCL17 expression appears to be restricted to mucosal tissues such as the tongue, stomach and lung, suggestive of specific roles at these locations. A putative CXCL17 receptor, GPR35 was reportedly identified and mice deficient in CXCL17 were generated and characterised. More recently, however, some apparent contradictions regarding aspects of CXCL17 biology have been raised by ourselves and others. Notably, GPR35 appears to be a receptor for the serotonin metabolite 5-hydroxyindoleacetic acid rather than for CXCL17 and modelling of CXCL17 using a variety of platforms fails to identify a chemokine-like fold. In this article, we summarize the discovery of CXCL17 and discuss key papers describing the subsequent characterisation of this protein. Ultimately, we pose the question, 'What defines a chemokine?' (185 words).

User Feedback on the Use of a Natural Language Processing Application to Screen for Suicide Risk in the Emergency Department.

Suicide is the 10th leading cause of death in the USA and globally. Despite decades of research, the ability to predict who will die by suicide is still no better than 50%. Traditional screening instruments have helped identify risk factors for suicide, but they have not provided accurate predictive power for reducing death rates. Over the past decade, natural language processing (NLP), a form of machine learning (ML), has been used to identify suicide risk by analyzing language data. Recent work has demonstrated the successful integration of a suicide risk screening interview to collect language data for NLP analysis from patients in two emergency departments (ED) of a large healthcare system. Results indicated that ML/NLP models performed well identifying patients that came to the ED for suicide risk. However, little is known about the clinician's perspective of how a qualitative brief interview suicide risk screening tool to collect language data for NLP integrates into an ED workflow. This report highlights the feedback and observations of patient experiences obtained from clinicians using brief suicide screening interviews. The investigator used an open-ended, narrative interview approach to inquire about the qualitative interview process. Three overarching themes were identified: behavioral health workflow, clinical implications of interview probes, and integration of an application into provider patient experience. Results suggest a brief, qualitative interview method was feasible, person-centered, and useful as a suicide risk detection approach.

Suicidal ideation in veterans enrolled in evidence-based psychotherapy for posttraumatic stress disorder.

Suicidal ideation (SI) is a highly prevalent public health issue in the veteran population and is increasingly common in veterans who are diagnosed with other mental health conditions, such as posttraumatic stress disorder (PTSD; U.S. Department of Veterans Affairs, 2020). The present study has an initial aim of examining changes in SI over treatment, and it is hypothesized that SI will decrease across PTSD treatments. A second aim is to examine the association of SI status with PTSD symptoms across treatment, and it is hypothesized that PTSD symptomatology will decrease at similar rates over the course of treatment for those who did and did not endorse SI at pretreatment. Participants included 717 (86.3% male) veterans who participated in outpatient treatment within a Veterans Affairs Post Traumatic Stress Disorder (VA PTSD) specialty clinic between July 2014 and December 2017. Descriptive analyses found that 37.2% of veterans endorsed SI at pretreatment, while 18.6% endorsed SI at posttreatment. The relationship between pre- and posttreatment SI was significant, χ²(1, N = 247) = 23.77, p < .001. A significant proportion of veterans who endorsed SI at pretreatment no longer endorsed SI at posttreatment (64.7%). There were no differences in changes in PTSD Checklist for DSM-5 (PCL-5) scores across treatment for those with and without SI at pretreatment. While those who endorsed SI at pretreatment had higher PCL-5 scores throughout treatment, they experienced a similar rate of improvement in symptoms as those without SI at pretreatment. This finding suggests that the presence of SI does not reduce the effectiveness of PTSD treatment. Limitations include the use of a single-item measure of SI, lack of adequate power to detect difference among treatments, and a cross-sectional design. Clinical and research implications are discussed. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Impact of residential PTSD treatment on suicide risk in veterans.

INTRODUCTION: In 2019, 17 veterans died by suicide every day. Various suicide prevention treatments have emerged, yet limited research has explored the impact of Post Traumatic Stress Disorder (PTSD) treatment on suicidal ideation and behaviors. METHODS: This study examines the impact of Cognitive Processing Therapy (CPT) on suicidal ideation among veterans in three residential PTSD programs (women's, men's, and PTSD/Traumatic Brain Injury). Interview and self-report data were collected from veterans (n = 446) throughout treatment. RESULTS: Over 50% of veterans reported current suicidal ideation and a history of suicide attempts prior to treatment. Variables that predicted change in suicidal ideation included prior suicide attempt (ß = 0.21, p = 0.022), change in CAPS-5 total score (ß = 0.28, p = 0.038), employment status (ß = -0.20, p = 0.035) and history of suicide attempt (ß = 0.25, p = 0.009). Those without a previous suicide attempt made greater gains in CPT treatment than those with a previous suicide attempt. CONCLUSION: Following 7 weeks of CPT residential treatment, a decrease in PTSD symptoms was significantly associated with a reduction in suicidal ideation. Implications are that CPT can reduce suicide risk in a variety of Veteran cohorts with differing trauma types.

Flavonols improve thermotolerance in tomato pollen during germination and tube elongation by maintaining ROS homeostasis.

Elevated temperatures impair pollen performance and reproductive success, resulting in lower crop yields. The Solanum lycopersicum anthocyanin reduced (are) mutant has a defect in the FLAVANONE 3 HYDROXYLASE (F3H) gene and impaired synthesis of flavonol antioxidants. We identified multiple aspects of pollen performance in are that were hypersensitive to elevated temperatures relative to the VF36 parental line, including heat-increased accumulation of reactive oxygen species (ROS). Transformation of are with an F3H transgene, or chemical complementation with flavonols, prevented temperature-dependent ROS accumulation in pollen and restored pollen performance to VF36 levels. Transformation of this F3H construct into VF36 (VF36-F3H-T3) prevented both temperature driven ROS increases and impaired pollen performance. RNA-Seq was performed at optimal and stress temperatures in are, VF36, and VF36-F3H-T3 at multiple timepoints across pollen tube emergence and elongation. All genotypes had increasing numbers of differentially expressed genes with duration of elevated temperature, with the largest number in are at all time points. These analyses also identified upregulated transcripts in are, relative to VF36, even at optimal temperatures, revealing a flavonol-regulated transcriptome. These findings suggest potential agricultural interventions to combat the negative effects of heat-induced ROS in pollen that leads to reproductive failure and crop loss.

Layered evolution of gene expression in "superfast" muscles for courtship.

Identifying the molecular process of complex trait evolution is a core goal of biology. However, pinpointing the specific context and timing of trait-associated changes within the molecular evolutionary history of an organism remains an elusive goal. We study this topic by exploring the molecular basis of elaborate courtship evolution, which represents an extraordinary example of trait innovation. Within the behaviorally diverse radiation of Central and South American manakin birds, species from two separate lineages beat their wings together using specialized "superfast" muscles to generate a "snap" that helps attract mates. Here, we develop an empirical approach to analyze phylogenetic lineage-specific shifts in gene expression in the key snap-performing muscle and then integrate these findings with comparative transcriptomic sequence analysis. We find that rapid wing displays are associated with changes to a wide range of molecular processes that underlie extreme muscle performance, including changes to calcium trafficking, myocyte homeostasis and metabolism, and hormone action. We furthermore show that these changes occur gradually in a layered manner across the species history, wherein which ancestral genetic changes to many of these molecular systems are built upon by later species-specific shifts that likely finalized the process of display performance adaptation. Our study demonstrates the potential for combining phylogenetic modeling of tissue-specific gene expression shifts with phylogenetic analysis of lineage-specific sequence changes to reveal holistic evolutionary histories of complex traits.

The Role of Streptococcal Cell-Envelope Proteases in Bacterial Evasion of the Innate Immune System.

Bacteria possess the ability to evolve varied and ingenious strategies to outwit the host immune system, instigating an evolutionary arms race. Proteases are amongst the many weapons employed by bacteria, which specifically cleave and neutralize key signalling molecules required for a coordinated immune response. In this article, we focus on a family of S8 subtilisin-like serine proteases expressed as cell-envelope proteases (CEPs) by group A and group B streptococci. Two of these proteases known as Streptococcus pyogenes CEP (SpyCEP) and C5a peptidase cleave the chemokine CXCL8 and the complement fragment C5a, respectively. Both CXCL8 and C5a are potent neutrophil-recruiting chemokines, and by neutralizing their activity, streptococci evade a key defence mechanism of innate immunity. We review the mechanisms by which CXCL8 and C5a recruit neutrophils and the characterization of SpyCEP and C5a peptidase, including both in vitro and in vivo studies. Recently described structural insights into the function of this CEP family are also discussed. We conclude by examining the progress of prototypic vaccines incorporating SpyCEP and C5a peptidase in their preparation. Since streptococci-producing SpyCEP and C5a peptidase are responsible for a considerable global disease burden, targeting these proteases by vaccination strategies or by small-molecule antagonists should provide protection from and promote the resolution of streptococcal infections.

A highly efficient method for the production and purification of recombinant human CXCL8.

Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.

Challenges and Opportunities in the Clinical Development of STING Agonists for Cancer Immunotherapy.

Immune checkpoint inhibitors (ICI) have revolutionised cancer therapy. However, they have been effective in only a small subset of patients and a principal mechanism underlying immune-refractoriness is a 'cold' tumour microenvironment, that is, lack of a T-cell-rich, spontaneously inflamed phenotype. As such, there is a demand to develop strategies to transform the tumour milieu of non-responsive patients to one supporting T-cell-based inflammation. The cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes (cGAS-STING) pathway is a fundamental regulator of innate immune sensing of cancer, with potential to enhance tumour rejection through the induction of a pro-inflammatory response dominated by Type I interferons. Recognition of these positive immune-modulatory properties has rapidly elevated the STING pathway as a putative target for immunotherapy, leading to a myriad of preclinical and clinical studies assessing natural and synthetic cyclic dinucleotides and non-nucleotidyl STING agonists. Despite pre-clinical evidence of efficacy, clinical translation has resulted into disappointingly modest efficacy. Poor pharmacokinetic and physiochemical properties of cyclic dinucleotides are key barriers to the development of STING agonists, most of which require intra-tumoral dosing. Development of systemically administered non-nucleotidyl STING agonists, or conjugation with liposomes, polymers and hydrogels may overcome pharmacokinetic limitations and improve drug delivery. In this review, we summarise the body of evidence supporting a synergistic role of STING agonists with currently approved ICI therapies and discuss whether, despite the numerous obstacles encountered to date, the clinical development of STING agonist as novel anti-cancer therapeutics may still hold the promise of broadening the reach of cancer immunotherapy.

Structure, dynamics and immunogenicity of a catalytically inactive CXC chemokine-degrading protease SpyCEP from Streptococcus pyogenes.

Over 18 million disease cases and half a million deaths worldwide are estimated to be caused annually by Group A Streptococcus. A vaccine to prevent GAS disease is urgently needed. SpyCEP (Streptococcus pyogenes Cell-Envelope Proteinase) is a surface-exposed serine protease that inactivates chemokines, impairing neutrophil recruitment and bacterial clearance, and has shown promising immunogenicity in preclinical models. Although SpyCEP structure has been partially characterized, a more complete and higher resolution understanding of its antigenic features would be desirable prior to large scale manufacturing. To address these gaps and facilitate development of this globally important vaccine, we performed immunogenicity studies with a safety-engineered SpyCEP mutant, and comprehensively characterized its structure by combining X-ray crystallography, NMR spectroscopy and molecular dynamics simulations. We found that the catalytically-inactive SpyCEP antigen conferred protection similar to wild-type SpyCEP in a mouse infection model. Further, a new higher-resolution crystal structure of the inactive SpyCEP mutant provided new insights into this large chemokine protease comprising nine domains derived from two non-covalently linked fragments. NMR spectroscopy and molecular simulation analyses revealed conformational flexibility that is likely important for optimal substrate recognition and overall function. These combined immunogenicity and structural data demonstrate that the full-length SpyCEP inactive mutant is a strong candidate human vaccine antigen. These findings show how a multi-disciplinary study was used to overcome obstacles in the development of a GAS vaccine, an approach applicable to other future vaccine programs. Moreover, the information provided may also facilitate the structure-based discovery of small-molecule therapeutics targeting SpyCEP protease inhibition.

The Catalytic-Dependent and -Independent Roles of Lsd1 and Lsd2 Lysine Demethylases in Heterochromatin Formation in Schizosaccharomyces pombe.

In eukaryotes, heterochromatin plays a critical role in organismal development and cell fate acquisition, through regulating gene expression. The evolutionarily conserved lysine-specific demethylases, Lsd1 and Lsd2, remove mono- and dimethylation on histone H3, serving complex roles in gene expression. In the fission yeast Schizosaccharomyces pombe, null mutations of Lsd1 and Lsd2 result in either severe growth defects or inviability, while catalytic inactivation causes minimal defects, indicating that Lsd1 and Lsd2 have essential functions beyond their known demethylase activity. Here, we show that catalytic mutants of Lsd1 or Lsd2 partially assemble functional heterochromatin at centromeres in RNAi-deficient cells, while the C-terminal truncated alleles of Lsd1 or Lsd2 exacerbate heterochromatin formation at all major heterochromatic regions, suggesting that Lsd1 and Lsd2 repress heterochromatic transcripts through mechanisms both dependent on and independent of their catalytic activities. Lsd1 and Lsd2 are also involved in the establishment and maintenance of heterochromatin. At constitutive heterochromatic regions, Lsd1 and Lsd2 regulate one another and cooperate with other histone modifiers, including the class II HDAC Clr3 and the Sirtuin family protein Sir2 for gene silencing, but not with the class I HDAC Clr6. Our findings explore the roles of lysine-specific demethylases in epigenetic gene silencing at heterochromatic regions.

A degradatory fate for CCR4 suggests a primary role in Th2 inflammation.

CCR4 is the sole receptor for the chemokines CCL22 and CCL17. Clinical studies of asthmatic airways have shown levels of both ligands and CCR4+ Th2 cells to be elevated, suggestive of a role in disease. Consequently, CCR4 has aroused much interest as a potential therapeutic target and an understanding of how its cell surface expression is regulated is highly desirable. To this end, receptor expression, receptor endocytosis, and chemotaxis were assessed using transfectants expressing CCR4, CCR4+ human T cell lines, and human Th2 cells polarized in vitro. CCL17 and CCL22 drove rapid endocytosis of CCR4 in a dose-dependent manner. Replenishment at the cell surface was slow and sensitive to cycloheximide, suggestive of de novo synthesis of CCR4. Constitutive CCR4 endocytosis was also observed, with the internalized CCR4 found to be significantly degraded over a 6-h incubation. Truncation of the CCR4 C-terminus by 40 amino acids had no effect on cell surface expression, but resulted in significant impairment of ligand-induced endocytosis. Consequently, migration to both CCL17 and CCL22 was significantly enhanced. In contrast, truncation of CCR4 did not impair constitutive endocytosis or degradation, suggesting the use of alternative receptor motifs in these processes. We conclude that CCR4 cell surface levels are tightly regulated, with a degradative fate for endocytosed receptor. We postulate that this strict control is desirable, given that Th2 cells recruited by CCR4 can induce the further expression of CCR4 ligands in a positive feedback loop, thereby enhancing allergic inflammation.

Pulmonary environmental cues drive group 2 innate lymphoid cell dynamics in mice and humans.

Group 2 innate lymphoid cells (ILC2s) are enriched in mucosal tissues (e.g., lung) and respond to epithelial cell-derived cytokines initiating type 2 inflammation. During inflammation, ILC2 numbers are increased in the lung. However, the mechanisms controlling ILC2 trafficking and motility within inflamed lungs remain unclear and are crucial for understanding ILC2 function in pulmonary immunity. Using several approaches, including lung intravital microscopy, we demonstrate that pulmonary ILC2s are highly dynamic, exhibit amoeboid-like movement, and aggregate in the lung peribronchial and perivascular spaces. They express distinct chemokine receptors, including CCR8, and actively home to CCL8 deposits located around the airway epithelium. Within lung tissue, ILC2s were particularly motile in extracellular matrix-enriched regions. We show that collagen-I drives ILC2 to markedly change their morphology by remodeling their actin cytoskeleton to promote environmental exploration critical for regulating eosinophilic inflammation. Our study provides previously unappreciated insights into ILC2 migratory patterns during inflammation and highlights the importance of environmental guidance cues in the lung in controlling ILC2 dynamics.

A Requirement for Neutrophil Glycosaminoglycans in Chemokine:Receptor Interactions Is Revealed by the Streptococcal Protease SpyCEP.

To evade the immune system, the lethal human pathogen Streptococcus pyogenes produces SpyCEP, an enzyme that cleaves the C-terminal α-helix of CXCL8, resulting in markedly impaired recruitment of neutrophils to sites of invasive infection. The basis for chemokine inactivation by SpyCEP is, however, poorly understood, as the core domain of CXCL8 known to interact with CXCL8 receptors is unaffected by enzymatic cleavage. We examined the in vitro migration of human neutrophils and observed that their ability to efficiently navigate a CXCL8 gradient was compromised following CXCL8 cleavage by SpyCEP. SpyCEP-mediated cleavage of CXCL8 also impaired CXCL8-induced migration of transfectants expressing the human chemokine receptors CXCR1 or CXCR2. Despite possessing an intact N terminus and preserved disulfide bonds, SpyCEP-cleaved CXCL8 had impaired binding to both CXCR1 and CXCR2, pointing to a requirement for the C-terminal α-helix. SpyCEP-cleaved CXCL8 had similarly impaired binding to the glycosaminoglycan heparin. Enzymatic removal of neutrophil glycosaminoglycans was observed to ablate neutrophil navigation of a CXCL8 gradient, whereas navigation of an fMLF gradient remained largely intact. We conclude, therefore, that SpyCEP cleavage of CXCL8 results in chemokine inactivation because of a requirement for glycosaminoglycan binding in productive chemokine:receptor interactions. This may inform strategies to inhibit the activity of SpyCEP, but may also influence future approaches to inhibit unwanted chemokine-induced inflammation.