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1.
Sci Adv ; 9(21): eadg5128, 2023 05 26.
Article in English | MEDLINE | ID: mdl-37235648

ABSTRACT

An intense, nonresolving airway inflammatory response leads to destructive lung disease in cystic fibrosis (CF). Dysregulation of macrophage immune function may be a key facet governing the progression of CF lung disease, but the underlying mechanisms are not fully understood. We used 5' end centered transcriptome sequencing to profile P. aeruginosa LPS-activated human CF macrophages, showing that CF and non-CF macrophages deploy substantially distinct transcriptional programs at baseline and following activation. This includes a significantly blunted type I IFN signaling response in activated patient cells relative to healthy controls that was reversible upon in vitro treatment with CFTR modulators in patient cells and by CRISPR-Cas9 gene editing to correct the F508del mutation in patient-derived iPSC macrophages. These findings illustrate a previously unidentified immune defect in human CF macrophages that is CFTR dependent and reversible with CFTR modulators, thus providing new avenues in the search for effective anti-inflammatory interventions in CF.


Subject(s)
Cystic Fibrosis , Humans , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Macrophages/metabolism , Signal Transduction , Mutation , Pseudomonas aeruginosa
2.
Sci Rep ; 13(1): 2271, 2023 02 08.
Article in English | MEDLINE | ID: mdl-36755116

ABSTRACT

The human skin barrier, a biological imperative, is impaired in inflammatory skin diseases such as atopic dermatitis (AD). Staphylococcus aureus is associated with AD lesions and contributes to pathological inflammation and further barrier impairment. S. aureus secretes extracellular proteases, such as V8 (or 'SspA'), which cleave extracellular proteins to reduce skin barrier. Previous studies demonstrated that the host defence peptide human beta-defensin 2 (HBD2) prevented V8-mediated damage. Here, the mechanism of HBD2-mediated barrier protection in vitro is examined. Application of exogenous HBD2 provided protection against V8, irrespective of timeline of application or native peptide folding, raising the prospect of simple peptide analogues as therapeutics. HBD2 treatment, in context of V8-mediated damage, modulated the proteomic/secretomic profiles of HaCaT cells, altering levels of specific extracellular matrix proteins, potentially recovering V8 damage. However, HBD2 alone did not substantially modulate cellular proteomic/secretomics profiles in the absence of damage, suggesting possible therapeutic targeting of lesion damage sites only. HBD2 did not show any direct protease inhibition or induce expression of known antiproteases, did not alter keratinocyte migration or proliferation, or form protective nanonet structures. These data validate the barrier-protective properties of HBD2 in vitro and establish key protein datasets for further targeted mechanistic analyses.


Subject(s)
Dermatitis, Atopic , beta-Defensins , Humans , beta-Defensins/pharmacology , beta-Defensins/metabolism , Staphylococcus aureus/metabolism , Proteomics , Skin/metabolism , Dermatitis, Atopic/drug therapy , Dermatitis, Atopic/pathology , Proteins
3.
PLoS Biol ; 20(8): e3001554, 2022 08.
Article in English | MEDLINE | ID: mdl-36026478

ABSTRACT

Multiple sclerosis (MS) is a highly prevalent demyelinating autoimmune condition; the mechanisms regulating its severity and progression are unclear. The IL-17-producing Th17 subset of T cells has been widely implicated in MS and in the mouse model, experimental autoimmune encephalomyelitis (EAE). However, the differentiation and regulation of Th17 cells during EAE remain incompletely understood. Although evidence is mounting that the antimicrobial peptide cathelicidin profoundly affects early T cell differentiation, no studies have looked at its role in longer-term T cell responses. Now, we report that cathelicidin drives severe EAE disease. It is released from neutrophils, microglia, and endothelial cells throughout disease; its interaction with T cells potentiates Th17 differentiation in lymph nodes and Th17 to exTh17 plasticity and IFN-γ production in the spinal cord. As a consequence, mice lacking cathelicidin are protected from severe EAE. In addition, we show that cathelicidin is produced by the same cell types in the active brain lesions in human MS disease. We propose that cathelicidin exposure results in highly activated, cytokine-producing T cells, which drive autoimmunity; this is a mechanism through which neutrophils amplify inflammation in the central nervous system.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Multiple Sclerosis , Animals , Antimicrobial Cationic Peptides , Antimicrobial Peptides , Cell Differentiation , Encephalomyelitis, Autoimmune, Experimental/pathology , Endothelial Cells/metabolism , Humans , Mice , Mice, Inbred C57BL , Th1 Cells/metabolism , Th1 Cells/pathology , Th17 Cells/metabolism , Cathelicidins
4.
Thorax ; 77(10): 960-967, 2022 10.
Article in English | MEDLINE | ID: mdl-34789559

ABSTRACT

INTRODUCTION: Bronchiectasis is characterised by excessive neutrophilic inflammation. Lipid mediators such as prostaglandins and leukotrienes have crucial roles in the inflammatory response. Further characterisation of these lipids and understanding the interplay of anti-inflammatory and proinflammatory lipid mediators could lead to the development of novel anti-inflammatory therapies for bronchiectasis. AIM: The aim of our study was to characterise the lipids obtained from serum and airways in patients with bronchiectasis in the stable state. METHODS: Six healthy volunteers, 10 patients with mild bronchiectasis, 15 with moderate bronchiectasis and 9 with severe bronchiectasis were recruited. All participants had 60 mL of blood taken and underwent a bronchoscopy while in the stable state. Lipidomics was done on serum and bronchoalveolar lavage fluid (BALF). RESULTS: In the stable state, in serum there were significantly higher levels of prostaglandin E2 (PGE2), 15-hydroxyeicosatetranoic acid (15-HETE) and leukotriene B4 (LTB4) in patients with moderate-severe disease compared with healthy volunteers. There was a significantly lower level of lipoxin A4 (LXA4) in severe bronchiectasis.In BALF, there were significantly higher levels of PGE2, 5-HETE, 15-HETE, 9-hydroxyoctadecadienoic acid and LTB4 in moderate-severe patients compared with healthy volunteers.In the stable state, there was a negative correlation of PGE2 and LTB4 with % predicted forced expiratory volume in 1 s and a positive correlation with antibiotic courses.LXA4 improved blood and airway neutrophil phagocytosis and bacterial killing in patients with bronchiectasis. Additionally LXA4 reduced neutrophil activation and degranulation. CONCLUSION: There is a dysregulation of lipid mediators in bronchiectasis with excess proinflammatory lipids. LXA4 improves the function of reprogrammed neutrophils. The therapeutic efficacy of LXA4 in bronchiectasis warrants further studies.


Subject(s)
Bronchiectasis , Lipoxins , Humans , Prostaglandins , Bronchiectasis/drug therapy , Dinoprostone , Neutrophils , Anti-Inflammatory Agents , Leukotriene B4
5.
J Cyst Fibros ; 21(2): 370-374, 2022 03.
Article in English | MEDLINE | ID: mdl-34799297

ABSTRACT

Macrophages represent prominent immune orchestrators of cystic fibrosis (CF) inflammation and, as such, are an ever-increasing focus of CF research with several reports of intrinsic immune dysfunction related to loss of CFTR activity in macrophages themselves. Animal models of CF have contributed, in no small part, to a deepening of our understanding of the pathophysiology of the disease and towards therapeutic development. A commonly-used animal model in CF research is the Cftrtm1Unc Tg(FABP-hCFTR) mouse, which displays gut-specific expression of a human CFTR transgene in order to rescue the high rate of early mortality in Cftr-null mice associated with severe intestinal obstruction. We find significant variation in the response to inflammatory challenge of patient macrophages and cells derived from the Cftrtm1Unc Tg(FABP-hCFTR) mouse and show that macrophages derived from this mouse exhibit aberrant expression of human CFTR. This may contribute to the absence of inflammatory changes in this model.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator , Cystic Fibrosis , Intestines/metabolism , Animals , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Disease Models, Animal , Humans , Macrophages , Mice , Mice, Inbred CFTR , Phenotype
6.
Int J Mol Sci ; 22(19)2021 Sep 23.
Article in English | MEDLINE | ID: mdl-34638591

ABSTRACT

Biofilm-related implant infections (BRII) are a disastrous complication of both elective and trauma orthopaedic surgery and occur when an implant becomes colonised by bacteria. The definitive treatment to eradicate the infections once a biofilm has established is surgical excision of the implant and thorough local debridement, but this carries a significant socioeconomic cost, the outcomes for the patient are often poor, and there is a significant risk of recurrence. Due to the large volumes of surgical procedures performed annually involving medical device implantation, both in orthopaedic surgery and healthcare in general, and with the incidence of implant-related infection being as high as 5%, interventions to prevent and treat BRII are a major focus of research. As such, innovation is progressing at a very fast pace; the aim of this study is to review the latest interventions for the prevention and treatment of BRII, with a particular focus on implant-related approaches.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Bacteria/drug effects , Biofilms/drug effects , Orthopedic Procedures/adverse effects , Postoperative Complications/microbiology , Prostheses and Implants/microbiology , Animals , Humans , Orthopedics/methods
7.
Front Immunol ; 12: 625922, 2021.
Article in English | MEDLINE | ID: mdl-34168640

ABSTRACT

Low density neutrophils (LDNs) are described in a number of inflammatory conditions, cancers and infections and associated with immunopathology, and a mechanistic role in disease. The role of LDNs at homeostasis in healthy individuals has not been investigated. We have developed an isolation protocol that generates high purity LDNs from healthy donors. Healthy LDNs were identical to healthy normal density neutrophils (NDNs), aside from reduced neutrophil extracellular trap formation. CD66b, CD16, CD15, CD10, CD54, CD62L, CXCR2, CD47 and CD11b were expressed at equivalent levels in healthy LDNs and NDNs and underwent apoptosis and ROS production interchangeably. Healthy LDNs had no differential effect on CD4+ or CD8+ T cell proliferation or IFNγ production compared with NDNs. LDNs were generated from healthy NDNs in vitro by activation with TNF, LPS or fMLF, suggesting a mechanism of LDN generation in disease however, we show neutrophilia in people with Cystic Fibrosis (CF) was not due to increased LDNs. LDNs are present in the neutrophil pool at homeostasis and have limited functional differences to NDNs. We conclude that increased LDN numbers in disease reflect the specific pathology or inflammatory environment and that neutrophil density alone is inadequate to classify discrete functional populations of neutrophils.


Subject(s)
Cell Separation , Flow Cytometry , Neutrophils/immunology , Antigens, CD/metabolism , Apoptosis , Case-Control Studies , Cell Proliferation , Cells, Cultured , Cellular Microenvironment , Centrifugation, Density Gradient , Cystic Fibrosis/immunology , Cystic Fibrosis/metabolism , Extracellular Traps/metabolism , Healthy Volunteers , Homeostasis , Humans , Interferon-gamma/metabolism , Leukocyte Count , Leukocyte Disorders/immunology , Leukocyte Disorders/metabolism , Lymphocyte Activation , Neutrophil Activation , Neutrophils/metabolism , Phenotype , Reactive Oxygen Species/metabolism , Receptors, Interleukin-8B/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
8.
Nat Commun ; 12(1): 1285, 2021 02 24.
Article in English | MEDLINE | ID: mdl-33627652

ABSTRACT

The host defence peptide cathelicidin (LL-37 in humans, mCRAMP in mice) is released from neutrophils by de-granulation, NETosis and necrotic death; it has potent anti-pathogen activity as well as being a broad immunomodulator. Here we report that cathelicidin is a powerful Th17 potentiator which enhances aryl hydrocarbon receptor (AHR) and RORγt expression, in a TGF-ß1-dependent manner. In the presence of TGF-ß1, cathelicidin enhanced SMAD2/3 and STAT3 phosphorylation, and profoundly suppressed IL-2 and T-bet, directing T cells away from Th1 and into a Th17 phenotype. Strikingly, Th17, but not Th1, cells were protected from apoptosis by cathelicidin. We show that cathelicidin is released by neutrophils in mouse lymph nodes and that cathelicidin-deficient mice display suppressed Th17 responses during inflammation, but not at steady state. We propose that the neutrophil cathelicidin is required for maximal Th17 differentiation, and that this is one method by which early neutrophilia directs subsequent adaptive immune responses.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Neutrophils/cytology , Neutrophils/drug effects , Th17 Cells/metabolism , Animals , Cell Differentiation/drug effects , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Phosphorylation/genetics , Phosphorylation/physiology , Pore Forming Cytotoxic Proteins/pharmacology , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Smad2 Protein/genetics , Smad2 Protein/metabolism , Smad3 Protein/genetics , Smad3 Protein/metabolism , Th1 Cells/cytology , Th1 Cells/drug effects , Th1 Cells/metabolism , Th17 Cells/cytology , Th17 Cells/drug effects , Cathelicidins
9.
Int Wound J ; 18(4): 448-456, 2021 Aug.
Article in English | MEDLINE | ID: mdl-33476481

ABSTRACT

Chronic wounds are a considerable health burden with high morbidity and poor rates of healing. Colonisation of chronic wounds by bacteria can be a significant factor in their poor healing rate. These bacteria can develop antibiotic resistance over time and can lead to wound infections, systemic illness, and occasionally amputation. When a large number of micro-organisms colonise wounds, they can lead to biofilm formation, which are self-perpetuating colonies of bacteria closed within an extracellular matrix, which are poorly penetrated by antibiotics. Platelet-rich plasma (PRP) is an autologous blood product rich in growth factors and cytokines that are involved in an inflammatory response. PRP can be injected or applied to a wound as a topical gel, and there is some interest regarding its antimicrobial properties and whether this can improve wound healing. This study aimed to evaluate the in vitro bacteriostatic effect of PRP. PRP was collected from healthy volunteers and processed into two preparations: activated PRP-activated with calcium chloride and ethanol; inactivated PRP. The activity of each preparation against Staphylococcus aureus and Staphylococcus epidermis was evaluated against a control by three experiments: bacterial kill assay to assess planktonic bacterial growth; plate colony assay to assess bacterial colony growth; and colony biofilm assay to assess biofilm growth. Compared with control, both preparations of PRP significantly inhibited growth of planktonic S aureus and S epidermis. Activated PRP reduced planktonic bacterial concentration more than inactivated PRP in both bacteria. Both PRP preparations significantly reduced bacterial colony counts for both bacteria when compared with control; however, there was no difference between the two. There was no difference found between biofilm growth in either PRP against control or against the other preparation. This study demonstrates that PRP does have an inhibitory effect on the growth of common wound pathogens. Activation may be an important factor in increasing the antimicrobial effect of PRP. However, we did not find evidence of an effect against more complex bacterial colonies.


Subject(s)
Platelet-Rich Plasma , Staphylococcal Infections , Wound Infection , Anti-Bacterial Agents/pharmacology , Humans , Staphylococcal Infections/drug therapy , Wound Healing , Wound Infection/drug therapy
10.
Eur Respir J ; 57(6)2021 06.
Article in English | MEDLINE | ID: mdl-33303535

ABSTRACT

Cystic fibrosis (CF) is a life-shortening, multi-organ, autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most prominent clinical manifestation in CF is the development of progressive lung disease characterised by an intense, chronic inflammatory airway response that culminates in respiratory failure and, ultimately, death. In recent years, a new class of therapeutics that have the potential to correct the underlying defect in CF, known as CFTR modulators, have revolutionised the field. Despite the exciting success of these drugs, their impact on airway inflammation, and its long-term consequences, remains undetermined. In addition, studies querying the absolute requirement for infection as a driver of CF inflammation have challenged the traditional consensus on CF pathogenesis, and also emphasise the need to prioritise complementary anti-inflammatory treatments in CF. Macrophages, often overlooked in CF research despite their integral role in other chronic inflammatory pathologies, have increasingly become recognised as key players in the initiation, perpetuation and resolution of CF lung inflammation, perhaps as a direct result of CFTR dysfunction. These findings suggest that macrophages may be an important target for novel anti-inflammatory interventional strategies to effectively treat CF lung function decline. This review will consider evidence for the efficacy of anti-inflammatory drugs in the treatment of CF, the potential role of macrophages, and the significance of targeting these pathways at a time when rectifying the basic defect in CF, through use of novel CFTR modulator therapies, is becoming increasingly viable.


Subject(s)
Cystic Fibrosis , Anti-Inflammatory Agents/therapeutic use , Cystic Fibrosis/drug therapy , Cystic Fibrosis Transmembrane Conductance Regulator , Humans , Inflammation/drug therapy , Macrophages
11.
Front Immunol ; 11: 1176, 2020.
Article in English | MEDLINE | ID: mdl-32595643

ABSTRACT

Defensins are short, rapidly evolving, cationic antimicrobial host defence peptides with a repertoire of functions, still incompletely realised, that extends beyond direct microbial killing. They are released or secreted at epithelial surfaces, and in some cases, from immune cells in response to infection and inflammation. Defensins have been described as endogenous alarmins, alerting the body to danger and responding to inflammatory signals by promoting both local innate and adaptive systemic immune responses. However, there is now increasing evidence that they exert variable control on the response to danger; creating a dichotomous response that can suppress inflammation in some circumstances but exacerbate the response to danger and damage in others and, at higher levels, lead to a cytotoxic effect. Focussing in this review on human ß-defensins, we discuss the evidence for their functions as proinflammatory, immune activators amplifying the response to infection or damage signals and/or as mediators of resolution of damage, contributing to a return to homeostasis. Finally, we consider their involvement in the development of autoimmune diseases.


Subject(s)
Autoimmune Diseases/immunology , Inflammation/immunology , beta-Defensins/immunology , Humans
12.
Placenta ; 93: 49-55, 2020 04.
Article in English | MEDLINE | ID: mdl-32250739

ABSTRACT

INTRODUCTION: Circulating DNA can be pro-inflammatory when detected by leukocytes via toll-like receptor 9 (TLR9). Cell-free fetal DNA (cff-DNA) of placental origin, circulates in pregnancy, and increased concentrations are seen in conditions associated with placental and maternal inflammation such as pre-eclampsia. However, whether cff-DNA is directly pro-inflammatory in pregnant women and what regulates cff-DNA levels in pregnancy are unknown. METHODS: Using a human term placental explant model, we examined whether induction of placental inflammation can promote cff-DNA release, and the capacity of this cff-DNA to stimulate peripheral blood mononuclear cells (PBMCs) from pregnant women. RESULTS: We demonstrate lipopolysaccharide (LPS)-mediated inflammation in placental explants and induced apoptosis after 24 h. However, this did not increase levels of cff-DNA generation compared to controls. Furthermore, the methylation status of the cff-DNA, was not altered by LPS-induced inflammation. Cff-DNA did not elicit production of inflammatory cytokines from PBMCs, in contrast to exposure to LPS or the TLR9 agonist CpG-ODN. Finally, we demonstrate that cff-DNA acquired directly from pregnant women did not differ in methylation status from placental extracted DNA, or from placental explant generated cell-free DNA, and that, unlike Escherichia coli DNA, this cff-DNA has a low level of unmethylated CpG sequences. DISCUSSION: Our data suggest that placental inflammation does not increase release of cff-DNA and that placental cff-DNA is not pro-inflammatory to circulating PBMCs. It thus seems unlikely that high levels of cff-DNA are either a direct consequence or cause of inflammation observed in obstetric complications.


Subject(s)
Cell-Free Nucleic Acids/physiology , Inflammation Mediators/physiology , Leukocytes, Mononuclear/metabolism , Placenta/metabolism , Adult , Apoptosis/drug effects , Cell-Free Nucleic Acids/metabolism , Cells, Cultured , Female , Humans , Inflammation/metabolism , Inflammation/pathology , Inflammation Mediators/metabolism , Leukocytes, Mononuclear/drug effects , Lipopolysaccharides/pharmacology , Placenta/drug effects , Placenta/pathology , Pregnancy
13.
BMJ Open ; 10(3): e035854, 2020 03 04.
Article in English | MEDLINE | ID: mdl-32139495

ABSTRACT

INTRODUCTION: Preterm birth is closely associated with altered brain development and is a leading cause of neurodevelopmental, cognitive and behavioural impairments across the life course. We aimed to investigate neuroanatomic variation and adverse outcomes associated with preterm birth by studying a cohort of preterm infants and controls born at term using brain MRI linked to biosamples and clinical, environmental and neuropsychological data. METHODS AND ANALYSIS: Theirworld Edinburgh Birth Cohort is a prospective longitudinal cohort study at the University of Edinburgh. We plan to recruit 300 infants born at <33 weeks of gestational age (GA) and 100 healthy control infants born after 37 weeks of GA. Multiple domains are assessed: maternal and infant clinical and demographic information; placental histology; immunoregulatory and trophic proteins in umbilical cord and neonatal blood; brain macrostructure and microstructure from structural and diffusion MRI (dMRI); DNA methylation; hypothalamic-pituitary-adrenal axis activity; social cognition, attention and processing speed from eye tracking during infancy and childhood; neurodevelopment; gut and respiratory microbiota; susceptibility to viral infections; and participant experience. Main analyses include creation of novel methods for extracting information from neonatal structural and dMRI, regression analyses of predictors of brain maldevelopment and neurocognitive outcome associated with preterm birth, and determination of the quantitative predictive performance of MRI and other early life factors for childhood outcome. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the National Research Ethics Service (NRES), South East Scotland Research Ethics Committee (NRES numbers 11/55/0061 and 13/SS/0143 (phase I) and 16/SS/0154 (phase II)), and NHS Lothian Research and Development (2016/0255). Results are disseminated through open access journals, scientific meetings, social media, newsletters anda study website (www.tebc.ed.ac.uk), and we engage with the University of Edinburgh public relations and media office to ensure maximum publicity and benefit.


Subject(s)
Child Development , Cognition , Premature Birth , Case-Control Studies , Child, Preschool , Developmental Disabilities/diagnosis , Female , Humans , Infant , Infant, Newborn , Infant, Premature , Longitudinal Studies , Male , Prospective Studies , Scotland , Surveys and Questionnaires
14.
Nat Rev Drug Discov ; 19(5): 311-332, 2020 05.
Article in English | MEDLINE | ID: mdl-32107480

ABSTRACT

Cationic host defence peptides (CHDP), also known as antimicrobial peptides, are naturally occurring peptides that can combat infections through their direct microbicidal properties and/or by influencing the host's immune responses. The unique ability of CHDP to control infections as well as resolve harmful inflammation has generated interest in harnessing the properties of these peptides to develop new therapies for infectious diseases, chronic inflammatory disorders and wound healing. Various strategies have been used to design synthetic optimized peptides, with negligible toxicity. Here, we focus on the progress made in understanding the scope of functions of CHDP and the emerging potential clinical applications of CHDP-based therapies.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Antimicrobial Cationic Peptides/therapeutic use , Communicable Diseases/drug therapy , Inflammation/drug therapy , Animals , Humans
15.
EFORT Open Rev ; 4(11): 633-639, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31754470

ABSTRACT

Prosthetic joint infection (PJI) is associated with poor clinical outcomes and is expensive to treat.Although uncommon overall (affecting between 0.5% and 2.2% of cases), PJI is one of the most commonly encountered complications of joint replacement and its incidence is increasing, putting a significant burden on healthcare systems.Once established, PJI is extremely difficult to eradicate as bacteria exist in biofilms which protect them from antibiotics and the host immune response.Improved understanding of the microbial pathology in PJI has generated potential new treatment strategies for prevention and eradication of biofilm associated infection including modification of implant surfaces to prevent adhesion of bacteria.Much research is currently ongoing looking at different implant surface coatings and modifications, and although most of this work has not translated into clinical medicine there has been some early clinical success. Cite this article: EFORT Open Rev 2019;4:633-639. DOI: 10.1302/2058-5241.4.180095.

16.
Oncoimmunology ; 8(8): 1608106, 2019.
Article in English | MEDLINE | ID: mdl-31413918

ABSTRACT

Immunization of patients with autologous, ex vivo matured dendritic cell (DC) preparations, in order to prime antitumor T-cell responses, is the focus of intense research. Despite progress and approval of clinical approaches, significant enhancement of these personalized immunotherapies is urgently needed to improve efficacy. We show that immunotherapeutic murine and human DC, generated in the presence of the antimicrobial host defense peptide LL-37, have dramatically enhanced expansion and differentiation of cells with key features of the critical CD103+/CD141+ DC subsets, including enhanced cross-presentation and co-stimulatory capacity, and upregulation of CCR7 with improved migratory capacity. These LL-37-DC enhanced proliferation, activation and cytokine production by CD8+ (but not CD4+) T cells in vitro and in vivo. Critically, tumor antigen-presenting LL-37-DC increased migration of primed, activated CD8+ T cells into established squamous cell carcinomas in mice, and resulted in tumor regression. This advance therefore has the potential to dramatically enhance DC immunotherapy protocols.

17.
Sci Rep ; 9(1): 7356, 2019 05 14.
Article in English | MEDLINE | ID: mdl-31089176

ABSTRACT

Preterm birth, defined as delivery before 37 weeks of gestation, is the leading cause of neonatal mortality and morbidity. Infection and inflammation are frequent antecedents of spontaneous preterm birth. Cathelicidin, an antimicrobial host defence peptide, is induced by infection and inflammation and although expressed in the reproductive tract and fetal tissues, its role in the pathogenesis of spontaneous preterm birth is unknown. Here we demonstrate that cathelicidin expression is increased at RNA and protein level in the mouse uterus in a model of inflammation-induced labour, where ultrasound guided intrauterine injection of lipopolysaccharide (LPS) at E17 stimulates preterm delivery within 24 hours. Cathelicidin-deficient (Camp-/-) mice are less susceptible to preterm delivery than wild type mice following intrauterine injection of 1 µg of LPS, and this is accompanied by a decrease in circulating IL-6, an inflammatory mediator implicated in the onset of labour. We also show that the proportion of cathelicidin expressing cells in the myometrium is higher in samples obtained from women in labour at term than pre-labour. Together, these data suggest that cathelicidin has roles in mediating pro-inflammatory responses in a murine model of inflammation-induced labour, and in human term labour.


Subject(s)
Antimicrobial Cationic Peptides/metabolism , Inflammation/immunology , Myometrium/pathology , Obstetric Labor, Premature/immunology , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/immunology , Cesarean Section , Disease Models, Animal , Female , Humans , Inflammation/blood , Inflammation/complications , Inflammation/pathology , Interleukin-6/blood , Interleukin-6/immunology , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/immunology , Mice , Mice, Knockout , Myometrium/immunology , Myometrium/surgery , Obstetric Labor, Premature/blood , Obstetric Labor, Premature/pathology , Pregnancy , RNA, Messenger/analysis , RNA, Messenger/metabolism , Cathelicidins
18.
PLoS Pathog ; 15(4): e1007694, 2019 04.
Article in English | MEDLINE | ID: mdl-30978238

ABSTRACT

Pulmonary infections are a major global cause of morbidity, exacerbated by an increasing threat from antibiotic-resistant pathogens. In this context, therapeutic interventions aimed at protectively modulating host responses, to enhance defence against infection, take on ever greater significance. Pseudomonas aeruginosa is an important multidrug-resistant, opportunistic respiratory pathogen, the clearance of which can be enhanced in vivo by the innate immune modulatory properties of antimicrobial host defence peptides from the cathelicidin family, including human LL-37. Initially described primarily as bactericidal agents, cathelicidins are now recognised as multifunctional antimicrobial immunomodulators, modifying host responses to pathogens, but the key mechanisms involved in these protective functions are not yet defined. We demonstrate that P. aeruginosa infection of airway epithelial cells promotes extensive infected cell internalisation of LL-37, in a manner that is dependent upon epithelial cell interaction with live bacteria, but does not require bacterial Type 3 Secretion System (T3SS). Internalised LL-37 acts as a second signal to induce inflammasome activation in airway epithelial cells, which, in contrast to myeloid cells, are relatively unresponsive to P. aeruginosa. We demonstrate that this is mechanistically dependent upon cathepsin B release, and NLRP3-dependent activation of caspase 1. These result in LL-37-mediated release of IL-1ß and IL-18 in a manner that is synergistic with P. aeruginosa infection, and can induce caspase 1-dependent death of infected epithelial cells, and promote neutrophil chemotaxis. We propose that cathelicidin can therefore act as a second signal, required by P. aeruginosa infected epithelial cells to promote an inflammasome-mediated altruistic cell death of infection-compromised epithelial cells and act as a "fire alarm" to enhance rapid escalation of protective inflammatory responses to an uncontrolled infection. Understanding this novel modulatory role for cathelicidins, has the potential to inform development of novel therapeutic strategies to antibiotic-resistant pathogens, harnessing innate immunity as a complementation or alternative to current interventions.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Cathelicidins/pharmacology , Epithelial Cells/immunology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pseudomonas Infections/immunology , Pseudomonas aeruginosa/immunology , Respiratory System/immunology , Animals , Caspase 1/metabolism , Cell Communication , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Immunity, Innate/drug effects , Immunity, Innate/immunology , Inflammasomes/drug effects , Inflammasomes/immunology , Inflammasomes/metabolism , Interleukin-18/genetics , Interleukin-18/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Male , Mice , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/metabolism , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Respiratory System/drug effects , Respiratory System/metabolism
19.
J Invest Dermatol ; 138(10): 2264-2274, 2018 10.
Article in English | MEDLINE | ID: mdl-29723492

ABSTRACT

Chronic wounds cause significant patient morbidity and mortality. A key factor in their etiology is microbial infection, yet skin host-microbiota interactions during wound repair remain poorly understood. Microbiome profiles of noninfected human chronic wounds are associated with subsequent healing outcome. Furthermore, poor clinical healing outcome was associated with increased local expression of the pattern recognition receptor NOD2. To investigate NOD2 function in the context of cutaneous healing, we treated mice with the NOD2 ligand muramyl dipeptide and analyzed wound repair parameters and expression of antimicrobial peptides. Muramyl dipeptide treatment of littermate controls significantly delayed wound repair associated with reduced re-epithelialization, heightened inflammation, and up-regulation of murine ß-defensins 1, 3, and particularly 14. We postulated that although murine ß-defensin 14 might affect local skin microbial communities, it may further affect other healing parameters. Indeed, exogenously administered murine ß-defensin 14 directly delayed mouse primary keratinocyte scratch wound closure in vitro. To further explore the role of murine ß-defensin 14 in wound repair, we used Defb14-/- mice and showed they had a global delay in healing in vivo, associated with alterations in wound microbiota. Taken together, these studies suggest a key role for NOD2-mediated regulation of local skin microbiota, which in turn affects chronic wound etiology.


Subject(s)
Microbiota/genetics , Nod2 Signaling Adaptor Protein/genetics , RNA/genetics , Up-Regulation , Wound Healing/genetics , Wounds and Injuries/genetics , beta-Defensins/genetics , Adult , Animals , Cells, Cultured , Disease Models, Animal , Female , Host Microbial Interactions , Humans , Keratinocytes/metabolism , Male , Mice , Mice, Inbred C57BL , Nod2 Signaling Adaptor Protein/metabolism , Real-Time Polymerase Chain Reaction , Transcriptional Activation , Wounds and Injuries/metabolism , Wounds and Injuries/pathology , beta-Defensins/metabolism
20.
Am J Respir Crit Care Med ; 198(7): 880-890, 2018 10 01.
Article in English | MEDLINE | ID: mdl-29733693

ABSTRACT

RATIONALE: Excessive neutrophilic airway inflammation is the central feature of bronchiectasis, but little is known about neutrophils in bronchiectasis. OBJECTIVES: To assess blood neutrophil phenotype in patients with bronchiectasis while stable and during exacerbations. METHODS: In the clinically stable arm of this study, there were eight healthy volunteers, eight patients with mild bronchiectasis, and eight patients with severe bronchiectasis. In addition, six patients with severe bronchiectasis were compared with six patients with community-acquired pneumonia at the start and end of an exacerbation. We assessed neutrophils for spontaneous apoptosis, cell surface marker expression, degranulation, reactive oxygen species generation, phagocytosis, and killing of Pseudomonas aeruginosa (PAO1). In addition, blood neutrophil function was compared with airway neutrophil function in bronchiectasis. MEASUREMENTS AND MAIN RESULTS: In stable bronchiectasis, compared with healthy volunteers, blood neutrophils had significantly prolonged viability, delayed apoptosis, increased CD62L shedding, upregulated CD11b expression, increased myeloperoxidase release, and impaired neutrophil phagocytosis and killing of PAO1. Bronchiectatic airway neutrophils had significantly lower bacterial phagocytosis and killing than their matched autologous blood neutrophils. Both blood and airway neutrophil phagocytosis and killing were impaired at the start of an exacerbation and improved following antibiotic treatment. In pneumonia, there was a significant improvement in phagocytosis and killing after treatment with antibiotics. During infections, there was no difference in phagocytosis, but there was significantly increased bacterial killing at the start and end of infection in pneumonia compared with bronchiectasis exacerbations. CONCLUSIONS: In bronchiectasis stable state, peripheral blood neutrophils are reprogrammed and have prolonged survival. This impairs their functional ability of bacterial phagocytosis and killing, thereby perpetuating the vicious circle in bronchiectasis.


Subject(s)
Apoptosis/physiology , Bronchiectasis/blood , Bronchiectasis/physiopathology , Neutrophils/cytology , Neutrophils/metabolism , Analysis of Variance , Bronchoscopy/methods , Case-Control Studies , Disease Progression , Female , Healthy Volunteers , Humans , Male , Middle Aged , Phagocytosis/physiology , Reactive Oxygen Species/metabolism , Reference Values , Sensitivity and Specificity , Severity of Illness Index
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