Publications by authors named "Nicolas Pionnier"

22 Publications

  • Page 1 of 1

CXCR4 signaling controls dendritic cell location and activation at steady-state and in inflammation.

Blood 2021 Jan 22. Epub 2021 Jan 22.

University Paris-Saclay, Clamart, France.

Dendritic cells (DCs) encompass several cell subsets that collaborate to initiate and regulate immune responses. Proper DC localization determines their function and requires the tightly controlled action of chemokine receptors. All DC subsets express CXCR4, but the genuine contribution of this receptor to their biology has been overlooked. We addressed this question using natural CXCR4 mutants resistant to CXCL12-induced desensitization and harboring a gain of function that cause the warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) Syndrome (WS), a rare immunodeficiency associated with high susceptibility to the pathogenesis of human papillomavirus (HPV). We report a reduction in the number of circulating plasmacytoid DCs (pDCs) in WHIM patients, whereas that of conventional DCs is preserved. This pattern was reproduced in an original mouse model of WS, enabling us to show that the circulating pDC defect can be corrected upon CXCR4 blockade and that pDC differentiation and function are preserved, despite CXCR4 dysfunction. We further identified proper CXCR4 signaling as a critical checkpoint for Langerhans-cell and DC migration from the skin to lymph nodes, with corollary alterations of their activation state and tissue inflammation in a model of HPV-induced dysplasia. Beyond providing new hypotheses to explain the susceptibility of WHIM patients to HPV pathogenesis, this study shows that proper CXCR4 signaling establishes a migration threshold that controls DC egress from CXCL12-containing environments and highlights the critical and subset-specific contribution of CXCR4 signal termination to DC biology.
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http://dx.doi.org/10.1182/blood.2020006675DOI Listing
January 2021

Tetracyclines improve experimental lymphatic filariasis pathology by disrupting interleukin-4 receptor-mediated lymphangiogenesis.

J Clin Invest 2021 Mar;131(5)

Centre for Drugs & Diagnostics, Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Lymphatic filariasis is the major global cause of nonhereditary lymphedema. We demonstrate that the filarial nematode Brugia malayi induced lymphatic remodeling and impaired lymphatic drainage following parasitism of limb lymphatics in a mouse model. Lymphatic insufficiency was associated with elevated circulating lymphangiogenic mediators, including vascular endothelial growth factor C. Lymphatic insufficiency was dependent on type 2 adaptive immunity, the interleukin-4 receptor, and recruitment of C-C chemokine receptor-2-positive monocytes and alternatively activated macrophages with a prolymphangiogenic phenotype. Oral treatments with second-generation tetracyclines improved lymphatic function, while other classes of antibiotic had no significant effect. Second-generation tetracyclines directly targeted lymphatic endothelial cell proliferation and modified type 2 prolymphangiogenic macrophage development. Doxycycline treatment impeded monocyte recruitment, inhibited polarization of alternatively activated macrophages, and suppressed T cell adaptive immune responses following infection. Our results determine a mechanism of action for the antimorbidity effects of doxycycline in filariasis and support clinical evaluation of second-generation tetracyclines as affordable, safe therapeutics for lymphedemas of chronic inflammatory origin.
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http://dx.doi.org/10.1172/JCI140853DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919730PMC
March 2021

Generation of Loa loa infective larvae by experimental infection of the vector, Chrysops silacea.

PLoS Negl Trop Dis 2020 08 17;14(8):e0008415. Epub 2020 Aug 17.

Parasites and Vector Biology research unit (PAVBRU), Department of Microbiology and Parasitology, University of Buea, Buea, Cameroon.

Basic and translational research on loiasis, a filarial nematode infection of medical importance, is impeded by a lack of suitable Loa loa infection models and techniques of obtaining and culturing life cycle stages. We describe the development of a new method for routine production of infective third-stage larvae (L3) of L. loa from the natural intermediate arthropod vector host, Chrysops silacea, following experimental infection with purified microfilariae. At 14-days post-infection of C. silacea, the fly survival rate was 43%. Survival was significantly higher in flies injected with 50 mf (55.2%) than those that received 100 mf (31.0%). However, yield per surviving fly and total yield of L3 was markedly higher in the group of flies inoculated with 100 mf (3474 vs 2462 L3 produced). The abdominal segment hosted the highest percentage recovery of L3 (47.7%) followed by head (34.5%) and thorax (17.9%). L. loa larval survival was higher than 90% after 30 days of in vitro culture. The in vitro moulting success rate to the L4 larval stage was 59.1%. After experimental infection of RAG2-/-IL-2γc-/-mice, the average L. loa juvenile adult worm recovery rate was 10.5% at 62 dpi. More than 87% of the worms were recovered from the muscles and subcutaneous tissues. Worms recovered measured an average 24.3 mm and 11.4 mm in length for females (n = 5) and males (n = 5), respectively. In conclusion, L. loa mf injected into C. silacea intrathoracically develop into infective larvae that remain viable and infective comparable to L3 obtained through natural feeding on the human host. This technique further advances the development of a full laboratory life cycle of L. loa where mf derived from experimentally-infected animals may be utilized to passage life cycle generations via intrathoracic injections of wild-caught vector hosts.
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http://dx.doi.org/10.1371/journal.pntd.0008415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470323PMC
August 2020

Eosinophil-Mediated Immune Control of Adult Filarial Nematode Infection Can Proceed in the Absence of IL-4 Receptor Signaling.

J Immunol 2020 08 22;205(3):731-740. Epub 2020 Jun 22.

Centre for Drugs and Diagnostics, Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, United Kingdom

Helminth infections are accompanied by eosinophilia in parasitized tissues. Eosinophils are effectors of immunity to tissue helminths. We previously reported that in the context of experimental filarial nematode infection, optimum tissue eosinophil recruitment was coordinated by local macrophage populations following IL-4R-dependent in situ proliferation and alternative activation. However, in the current study, we identify that control of chronic adult filarial worm infection is evident in IL-4Rα-deficient (IL-4Rα) mice, whereby the majority of infections do not achieve patency. An associated residual eosinophilia was apparent in infected IL-4Rα mice. By treating IL-4Rα mice serially with anti-CCR3 Ab or introducing a compound deficiency in CCR3 within IL-4Rα mice, residual eosinophilia was ablated, and susceptibility to chronic adult infection was established, promoting a functional role for CCR3-dependent eosinophil influx in immune control in the absence of IL-4/IL-13-dependent immune mechanisms. We investigated additional cytokine signals involved in residual eosinophilia in the absence IL-4Rα signaling and defined that IL-4Rα/IL-5 double-knockout mice displayed significant eosinophil deficiency compared with IL-4Rα mice and were susceptible to chronic fecund adult filarial infections. Contrastingly, there was no evidence that either IL-4R-dependent or IL-4R-independent/CCR3/IL-5-dependent immunity influenced microfilarial loads in the blood. Our data demonstrate multiplicity of Th2-cytokine control of eosinophil tissue recruitment during chronic filarial infection and that IL-4R-independent/IL-5- and CCR3-dependent pathways are sufficient to control filarial adult infection via an eosinophil-dependent effector response prior to patency.
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http://dx.doi.org/10.4049/jimmunol.1901244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372315PMC
August 2020

The insufficiency of circulating miRNA and DNA as diagnostic tools or as biomarkers of treatment efficacy for Onchocerca volvulus.

Sci Rep 2020 04 21;10(1):6672. Epub 2020 Apr 21.

Centre for Drugs and Diagnostics, Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Skin snip evaluation for onchocerciasis has insufficient sensitivity when skin microfilarial (mf) densities are low, such as following ivermectin treatment. Mf density is suitable for assessing microfilaricidal efficacy but only serves as an indirect indicator of macrofilaricidal activity. We assessed circulating nucleic acids from Onchocerca volvulus as an alternative to skin snips. We screened a plasma sample set of infected individuals followed up at four, 12 and 21 months after microfilaricidal (ivermectin, n = four), macrofilaricidal (doxycycline, n = nine), or combination treatment (n = five). Two parasite-derived miRNAs, cel-miR-71-5p and bma-lin-4, and O-150 repeat DNA were assessed. Highly abundant DNA repeat families identified in the O. volvulus genome were also evaluated. miRNAs were detected in two of 72 plasma samples (2.8%) and two of 47 samples (4.3%) with microfilaridermia using RT-qPCR. O-150 DNA was detected in eight (44.4%) baseline samples by qPCR and the number of positives declined post-treatment. One doxycycline-treated individual remained O-150 positive. However, only 11 (23.4%) samples with microfilaridermia were qPCR-positive. Analysis by qPCR showed novel DNA repeat families were comparatively less abundant than the O-150 repeat. Circulating parasite-derived nucleic acids are therefore insufficient as diagnostic tools or as biomarkers of treatment efficacy for O. volvulus.
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http://dx.doi.org/10.1038/s41598-020-63249-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174290PMC
April 2020

Discovery of short-course antiwolbachial quinazolines for elimination of filarial worm infections.

Sci Transl Med 2019 05;11(491)

Calibr at Scripps Research, La Jolla, CA 92037, USA.

Parasitic filarial nematodes cause debilitating infections in people in resource-limited countries. A clinically validated approach to eliminating worms uses a 4- to 6-week course of doxycycline that targets , a bacterial endosymbiont required for worm viability and reproduction. However, the prolonged length of therapy and contraindication in children and pregnant women have slowed adoption of this treatment. Here, we describe discovery and optimization of quinazolines CBR417 and CBR490 that, with a single dose, achieve >99% elimination of in the in vivo filarial infection model. The efficacious quinazoline series was identified by pairing a primary cell-based high-content imaging screen with an orthogonal ex vivo validation assay to rapidly quantify elimination in filarial ovaries. We screened 300,368 small molecules in the primary assay and identified 288 potent and selective hits. Of 134 primary hits tested, only 23.9% were active in the worm-based validation assay, 8 of which contained a quinazoline heterocycle core. Medicinal chemistry optimization generated quinazolines with excellent pharmacokinetic profiles in mice. Potent antiwolbachial activity was confirmed in , , and in vivo preclinical models of filarial disease and in vitro selectivity against (a safety concern in endemic areas). The favorable efficacy and in vitro safety profiles of CBR490 and CBR417 further support these as clinical candidates for treatment of filarial infections.
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http://dx.doi.org/10.1126/scitranslmed.aav3523DOI Listing
May 2019

Mouse models of Loa loa.

Nat Commun 2019 03 29;10(1):1429. Epub 2019 Mar 29.

Centre for Drugs and Diagnostics Research, Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.

Elimination of the helminth disease, river blindness, remains challenging due to ivermectin treatment-associated adverse reactions in loiasis co-infected patients. Here, we address a deficit in preclinical research tools for filarial translational research by developing Loa loa mouse infection models. We demonstrate that adult Loa loa worms in subcutaneous tissues, circulating microfilariae (mf) and presence of filarial biomarkers in sera occur following experimental infections of lymphopenic mice deficient in interleukin (IL)-2/7 gamma-chain signaling. A microfilaraemic infection model is also achievable, utilizing immune-competent or -deficient mice infused with purified Loa mf. Ivermectin but not benzimidazole treatments induce rapid decline (>90%) in parasitaemias in microfilaraemic mice. We identify up-regulation of inflammatory markers associated with allergic type-2 immune responses and eosinophilia post-ivermectin treatment. Thus, we provide validation of murine research models to identify loiasis biomarkers, to counter-screen candidate river blindness cures and to interrogate the inflammatory etiology of loiasis ivermectin-associated adverse reactions.
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http://dx.doi.org/10.1038/s41467-019-09442-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6441053PMC
March 2019

Preclinical development of an oral anti- macrolide drug for the treatment of lymphatic filariasis and onchocerciasis.

Sci Transl Med 2019 03;11(483)

Centre for Drugs and Diagnostics, Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK.

There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis ( and ) and onchocerciasis (). A 1- or 2-week course of oral A-1535469 or A-1574083 provided >90% depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations >10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti- macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis.
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http://dx.doi.org/10.1126/scitranslmed.aau2086DOI Listing
March 2019

Short-course, oral flubendazole does not mediate significant efficacy against Onchocerca adult male worms or Brugia microfilariae in murine infection models.

PLoS Negl Trop Dis 2019 01 16;13(1):e0006356. Epub 2019 Jan 16.

Department of Parasitology and Research Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, United Kingdom.

The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with >80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile.
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http://dx.doi.org/10.1371/journal.pntd.0006356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334903PMC
January 2019

The effect of the orientation and the height of periodic sub-micrometric texturing on dropwise condensation.

J Colloid Interface Sci 2018 Sep 30;526:184-193. Epub 2018 Apr 30.

Université de Lyon, Ecole Centrale de Lyon, Laboratoire de Tribologie et Dynamique des Systèmes, UMR 5513, F-69131 Ecully, France. Electronic address:

Controlling condensation conditions by surface topography is of prime interest. The aim of this work is to investigate the behavior of water droplets condensing on oriented sub-micrometric structures representing ripples with wavelengths around 800 nm. Droplet behavior was studied on different ripples heights and on untextured surfaces. It was specifically looked at how the presence of ripples creates geometrical confinement, and how that influences the deformation and the orientation of single droplets. Results show that the condensed droplets follow the orientation of textured features, especially with high structures height (150 nm). This is shown by the decreasing of droplet roundness with ripples height. The relative number of circular droplets (roundness near to 1) is around 0.6 for 70 nm high ripples and decrease to around 0.2 for 150 nm high ripples. The corresponding relative number on untextured surface is around 0.5. A mechanism, based on droplets pinning and hysteresis, is proposed to explain the influence of the ripples orientation in a vertical plane, onto the droplet deformation during coalescence step. Finally, the presence of ripples is shown to barely impact breath figure dynamics. Number of droplets and mean droplet radius for the textured and untextured surfaces present a comparable evolution.
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http://dx.doi.org/10.1016/j.jcis.2018.04.043DOI Listing
September 2018

Interleukin-4 activated macrophages mediate immunity to filarial helminth infection by sustaining CCR3-dependent eosinophilia.

PLoS Pathog 2018 03 16;14(3):e1006949. Epub 2018 Mar 16.

Research Centre for Drugs & Diagnostics, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Eosinophils are effectors in immunity to tissue helminths but also induce allergic immunopathology. Mechanisms of eosinophilia in non-mucosal tissues during infection remain unresolved. Here we identify a pivotal function of tissue macrophages (Mϕ) in eosinophil anti-helminth immunity using a BALB/c mouse intra-peritoneal Brugia malayi filarial infection model. Eosinophilia, via C-C motif chemokine receptor (CCR)3, was necessary for immunity as CCR3 and eosinophil impairments rendered mice susceptible to chronic filarial infection. Post-infection, peritoneal Mϕ populations proliferated and became alternatively-activated (AAMϕ). Filarial AAMϕ development required adaptive immunity and interleukin-4 receptor-alpha. Depletion of Mϕ prior to infection suppressed eosinophilia and facilitated worm survival. Add back of filarial AAMϕ in Mϕ-depleted mice recapitulated a vigorous eosinophilia. Transfer of filarial AAMϕ into Severe-Combined Immune Deficient mice mediated immunological resistance in an eosinophil-dependent manner. Exogenous IL-4 delivery recapitulated tissue AAMϕ expansions, sustained eosinophilia and mediated immunological resistance in Mϕ-intact SCID mice. Co-culturing Brugia with filarial AAMϕ and/or filarial-recruited eosinophils confirmed eosinophils as the larvicidal cell type. Our data demonstrates that IL-4/IL-4Rα activated AAMϕ orchestrate eosinophil immunity to filarial tissue helminth infection.
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http://dx.doi.org/10.1371/journal.ppat.1006949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874077PMC
March 2018

Migratory phase of Litomosoides sigmodontis filarial infective larvae is associated with pathology and transient increase of S100A9 expressing neutrophils in the lung.

PLoS Negl Trop Dis 2017 May 9;11(5):e0005596. Epub 2017 May 9.

Unité Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR 7245), Sorbonne Universités, Muséum national d'Histoire naturelle, CNRS, Paris, France.

Filarial infections are tropical diseases caused by nematodes of the Onchocercidae family such as Mansonella perstans. The infective larvae (L3) are transmitted into the skin of vertebrate hosts by blood-feeding vectors. Many filarial species settle in the serous cavities including M. perstans in humans and L. sigmodontis, a well-established model of filariasis in mice. L. sigmodontis L3 migrate to the pleural cavity where they moult into L4 around day 9 and into male and female adult worms around day 30. Little is known of the early phase of the parasite life cycle, after the L3 is inoculated in the dermis by the vector and enters the afferent lymphatic vessels and before the moulting processes in the pleural cavity. Here we reveal a pulmonary phase associated with lung damage characterized by haemorrhages and granulomas suggesting L3 reach the lung via pulmonary capillaries and damage the endothelium and parenchyma by crossing them to enter the pleural cavity. This study also provides evidence for a transient inflammation in the lung characterized by a very early recruitment of neutrophils associated with high expression levels of S100A8 and S100A9 proteins.
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http://dx.doi.org/10.1371/journal.pntd.0005596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5438187PMC
May 2017

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

Sci Rep 2017 03 16;7(1):210. Epub 2017 Mar 16.

Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK.

Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18-24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.
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http://dx.doi.org/10.1038/s41598-017-00322-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428297PMC
March 2017

Wolbachia endosymbionts induce neutrophil extracellular trap formation in human onchocerciasis.

Sci Rep 2016 10 18;6:35559. Epub 2016 Oct 18.

Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place L3 5QA, Liverpool, UK.

The endosymbiotic bacteria, Wolbachia, induce neutrophilic responses to the human helminth pathogen Onchocerca volvulus. The formation of Neutrophil Extracellular Traps (NETs), has been implicated in anti-microbial defence, but has not been identified in human helminth infection. Here, we demonstrate NETs formation in human onchocerciasis. Extracellular NETs and neutrophils were visualised around O. volvulus in nodules excised from untreated patients but not in nodules from patients treated with the anti-Wolbachia drug, doxycycline. Whole Wolbachia or microspheres coated with a synthetic Wolbachia lipopeptide (WoLP) of the major nematode Wolbachia TLR2/6 ligand, peptidoglycan associated lipoprotein, induced NETosis in human neutrophils in vitro. TLR6 dependency of Wolbachia and WoLP NETosis was demonstrated using purified neutrophils from TLR6 deficient mice. Thus, we demonstrate for the first time that NETosis occurs during natural human helminth infection and demonstrate a mechanism of NETosis induction via Wolbachia endobacteria and direct ligation of Wolbachia lipoprotein by neutrophil TLR2/6.
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http://dx.doi.org/10.1038/srep35559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067710PMC
October 2016

Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

PLoS Negl Trop Dis 2016 Apr 25;10(4):e0004605. Epub 2016 Apr 25.

Unité Molécules de Communication et Adaptation des Microorganismes (MCAM, UMR 7245), Sorbonne Universités, Muséum National d'Histoire Naturelle, CNRS; CP52, Paris, France.

Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis.
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http://dx.doi.org/10.1371/journal.pntd.0004605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4844152PMC
April 2016

Differential effects of alloherpesvirus CyHV-3 and rhabdovirus SVCV on apoptosis in fish cells.

Vet Microbiol 2015 Mar 23;176(1-2):19-31. Epub 2014 Dec 23.

Institute of Science and Technology in Medicine, School of Life Sciences, Keele University, ST5 5BG Keele, United Kingdom. Electronic address:

Whilst Herpesviridae, which infect higher vertebrates, actively influence host immune responses to ensure viral replication, it is mostly unknown if Alloherpesviridae, which infect lower vertebrates, possess similar abilities. An important antiviral response is clearance of infected cells via apoptosis, which in mammals influences the outcome of infection. Here, we utilise common carp infected with CyHV-3 to determine the effect on the expression of genes encoding apoptosis-related proteins (p53, Caspase 9, Apaf-1, IAP, iNOS) in the pronephros, spleen and gills. The influence of CyHV-3 on CCB cells was also studied and compared to SVCV (a rhabdovirus) which induces apoptosis in carp cell lines. Although CyHV-3 induced iNOS expression in vivo, significant induction of the genetic apoptosis pathway was only seen in the pronephros. In vitro CyHV-3 did not induce apoptosis or apoptosis-related expression whilst SVCV did stimulate apoptosis. This suggests that CyHV-3 possesses mechanisms similar to herpesviruses of higher vertebrates to inhibit the antiviral apoptotic process.
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http://dx.doi.org/10.1016/j.vetmic.2014.12.012DOI Listing
March 2015

C-reactive protein and complement as acute phase reactants in common carp Cyprinus carpio during CyHV-3 infection.

Dis Aquat Organ 2014 Jul;109(3):187-99

Institute of Science and Technology in Medicine, School of Life Sciences, Keele University, ST5 5BG Keele, UK.

Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a highly virulent and lethal disease of common carp Cyprinus carpio and its ornamental koi varieties. However, specific knowledge about immune mechanisms behind the infection process is very limited. We aimed to evaluate the effect of the CyHV-3 infection on the profile of 2 major components of the common carp immune acute phase response: the C-reactive protein (CRP) and the complement system. Common carp were infected with CyHV-3 by bath immersion. Fish were sampled before the infection and at 6, 12, 24, 72, 120 and 336 h post-infection for serum and head kidney, liver, gill and spleen tissues. CRP levels and complement activity were determined from the serum, whereas CRP- and complement-related genes (crp1, crp2, c1rs, bf/c2, c3, masp2) expression profiles were analysed in the tissues by quantitative PCR. Both CRP levels and complement activity increased significantly up to 10- and 3-fold, respectively, in the serum of infected fish during the challenge. Analysis revealed distinct organ- and time-dependent expression profile patterns for all selected genes. These results suggest that CRP and complement behave as acute phase reactants to CyHV-3 infection in common carp with an organ- and time-dependent response.
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http://dx.doi.org/10.3354/dao02727DOI Listing
July 2014

Feeding common carp Cyprinus carpio with β-glucan supplemented diet stimulates C-reactive protein and complement immune acute phase responses following PAMPs injection.

Fish Shellfish Immunol 2014 Aug 14;39(2):285-95. Epub 2014 May 14.

Institute of Science and Technology in Medicine, School of Life Sciences, Keele University, ST5 5BG Keele, United Kingdom. Electronic address:

The effect of β-glucan as a feed additive on the serum and gene profile of C-reactive protein (CRP) and complement acute phase responses was ascertained in common carp Cyprinus carpio. In addition effects of subsequent intraperitoneal injections of pathogen-associated molecular patterns (PAMPs), i.e. LPS or poly(I:C), to mimic bacterial or viral infection respectively, were studied. Carp were first orally fed with β-glucan (MacroGard®) with a daily β-glucan intake of 6 mg per kg body weight or with control food for 25 days and then injected with PBS containing either LPS (4 mg/kg) or poly(I:C) (5 mg/kg) or PBS alone. Fish were sampled during the 25 days of the feeding period and up to 7 days post-PAMPs injections for serum and liver, head kidney and mid-gut tissues. Oral administration of β-glucan for 25 days significantly increased serum CRP levels and alternative complement activity (ACP). In addition, the subsequent LPS and poly(I:C) challenges significantly affected CRP and complement related gene expression profiles (crp1, crp2, c1r/s, bf/c2, c3 and masp2), with the greatest effects observed in the β-glucan fed fish. However, in fish fed β-glucan the PAMPs injections had less effects on CRP levels and complement activity in the serum than in control fed fish, suggesting that the 25 days of β-glucan immunostimulation was sufficient enough to reduce the effects of LPS and poly(I:C) injections. Results suggest that MacroGard® stimulated CRP and complement responses to PAMPs immunological challenges in common carp thus highlighting the beneficial β-glucan immunostimulant properties.
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http://dx.doi.org/10.1016/j.fsi.2014.05.008DOI Listing
August 2014

β-Glucan-supplemented diets increase poly(I:C)-induced gene expression of Mx, possibly via Tlr3-mediated recognition mechanism in common carp (Cyprinus carpio).

Fish Shellfish Immunol 2014 Feb 25;36(2):494-502. Epub 2013 Dec 25.

School of Life Sciences, Keele University, Keele, Staffordshire ST5 5BG, United Kingdom. Electronic address:

We have previously observed that in common carp (Cyprinus carpio), administration of β-glucan (MacroGard®) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection, particularly in the gut. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through β-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by β-glucans in fish, MacroGard® was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly(I:C)), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response. Subsequently, a set of immune related genes, including mx, were analysed by real-time PCR on liver, spleen, head kidney and mid gut tissues. Results obtained confirmed that treatment with β-glucan alone generally down-regulated the mRNA expression of selected cytokines when compared to untreated fish, while mx gene expression remained stable or was slightly up-regulated. Injection with poly(I:C) induced a similar down-regulated gene expression pattern for cytokines in samples from β-glucan fed fish. In contrast, poly(I:C) injection markedly increased mx gene expression in samples from β-glucan fed fish but hardly in samples from fish fed control feed. In an attempt to explain the high induction of mx, we studied Toll-like receptor 3 (TLR3) gene expression in these carp. TLR3 is a prototypical pattern recognition receptor considered important for the binding of viral double-stranded RNA and triggering of a type-I IFN response. Through genome data mining, two sequences for carp tlr3 were retrieved (tlr3.1 and tlr3.2) and characterized. Constitutive gene expression of both tlr3.1 and tlr3.2 was detected by real-time PCR in cDNA of all analysed carp organs. Strikingly, 25 days after β-glucan feeding, very high levels of tlr3.1 gene expression were observed in all analysed organs, with the exception of the liver. Our data suggest that β-glucan-mediated protection against viral diseases could be due to an increased Tlr3-mediated recognition of ligands, resulting in an increased antiviral activity of Mx.
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http://dx.doi.org/10.1016/j.fsi.2013.12.005DOI Listing
February 2014

Dietary β-glucan stimulate complement and C-reactive protein acute phase responses in common carp (Cyprinus carpio) during an Aeromonas salmonicida infection.

Fish Shellfish Immunol 2013 Mar 2;34(3):819-31. Epub 2013 Jan 2.

Institute of Science and Technology in Medicine, School of Life Sciences, Keele University, ST5 5BG Keele, United Kingdom.

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard® stimulated CRP and complement responses to A. salmonicida infection in common carp.
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http://dx.doi.org/10.1016/j.fsi.2012.12.017DOI Listing
March 2013

Reduced inflammatory response to Aeromonas salmonicida infection in common carp (Cyprinus carpio L.) fed with β-glucan supplements.

Fish Shellfish Immunol 2012 Jun 2;32(6):1051-7. Epub 2012 Mar 2.

School of Life Sciences, Keele University, Keele, Staffordshire, United Kingdom.

The objective of the present study was to determine the action of β-glucans as feed additives on the gene expression profile of some inflammatory-related cytokines from common carp (Cyprinus carpio L.) during the early stages of a non-lethal bacterial infection with Aeromonas salmonicida. β-glucan (MacroGard(®)), was administered daily to carp (6 mg per kg body weight) in the form of supplemented commercial food pellets for 14 days prior to infection. Control and treated fish were then intraperitoneally injected with PBS or 4×10(8) bacteria per fish and were sampled at time 0 and 6h, 12h, 1 day, 3 days and 5 days post-injection. Head kidney and gut were collected and the gene expression patterns for tnfα1, tnfα2, il1β, il6 and il10 were analyzed by quantitative PCR. Results obtained showed that treatment with β-glucans generally down-regulated the expression of all measured genes when compared to their corresponding controls. After injection, highest changes in the gene expression levels were obtained at 6h; particularly, in head kidney there was higher up-regulation of tnfa1 and tnfa2 in infected fish fed β-glucans in comparison to control feed; however, in gut there was a significant down-regulation of tnfα1, tnfα2, il1β and il6 in infected fish fed β-glucans. Analysis of carp specific antibodies against A. salmonicida 30 days after injection revealed their levels were reduced in the infected β-glucan group. In conclusion, a diet supplemented with β-glucan (MacroGard(®)) reduced the gene expression levels of some inflammation-related cytokines in common carp. Such a response appears to be dependent of organ studied and therefore the immunostimulant may be preventing an acute and potential dangerous response in gut, whilst enhancing the inflammatory response in head kidney when exposed to A. salmonicida.
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http://dx.doi.org/10.1016/j.fsi.2012.02.028DOI Listing
June 2012