Publications by authors named "Marianna Parlato"

29 Publications

  • Page 1 of 1

Duplication of the IL2RA locus causes excessive IL-2 signaling and may predispose to very early onset colitis.

Mucosal Immunol 2021 Jul 5. Epub 2021 Jul 5.

Laboratory of Pediatrics, division Gastroenterology and Nutrition, Erasmus University Medical Center-Sophia Children's Hospital, Rotterdam, the Netherlands.

Single genetic mutations predispose to very early onset inflammatory bowel disease (VEO-IBD). Here, we identify a de novo duplication of the 10p15.1 chromosomal region, including the IL2RA locus, in a 2-year-old girl with treatment-resistant pancolitis that was brought into remission by colectomy. Strikingly, after colectomy while the patient was in clinical remission and without medication, the peripheral blood CD4:CD8 ratio was constitutively high and CD25 expression was increased on circulating effector memory, Foxp3, and Foxp3 CD4 T cells compared to healthy controls. This high CD25 expression increased IL-2 signaling, potentiating CD4 T-cell-derived IFNγ secretion after T-cell receptor (TCR) stimulation. Restoring CD25 expression using the JAK1/3-inhibitor tofacitinib controlled TCR-induced IFNγ secretion in vitro. As diseased colonic tissue, but not the unaffected duodenum, contained mainly CD4 T cells with a prominent IFNγ-signature, we hypothesize that local microbial stimulation may have initiated colonic disease. Overall, we identify that duplication of the IL2RA locus can associate with VEO-IBD and suggest that increased IL-2 signaling predisposes to colonic intestinal inflammation.
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http://dx.doi.org/10.1038/s41385-021-00423-5DOI Listing
July 2021

Bi-allelic variants in IPO8 cause a connective tissue disorder associated with cardiovascular defects, skeletal abnormalities, and immune dysregulation.

Am J Hum Genet 2021 06 18;108(6):1126-1137. Epub 2021 May 18.

Victorian Clinical Genetics Services, Murdoch Children's Research Institute, Parkville 3052, Melbourne, VIC, Australia; Department of Paediatrics, The University of Melbourne, 3010 Parkville, Melbourne, VIC, Australia.

Dysregulated transforming growth factor TGF-β signaling underlies the pathogenesis of genetic disorders affecting the connective tissue such as Loeys-Dietz syndrome. Here, we report 12 individuals with bi-allelic loss-of-function variants in IPO8 who presented with a syndromic association characterized by cardio-vascular anomalies, joint hyperlaxity, and various degree of dysmorphic features and developmental delay as well as immune dysregulation; the individuals were from nine unrelated families. Importin 8 belongs to the karyopherin family of nuclear transport receptors and was previously shown to mediate TGF-β-dependent SMADs trafficking to the nucleus in vitro. The important in vivo role of IPO8 in pSMAD nuclear translocation was demonstrated by CRISPR/Cas9-mediated inactivation in zebrafish. Consistent with IPO8's role in BMP/TGF-β signaling, ipo8 zebrafish presented mild to severe dorso-ventral patterning defects during early embryonic development. Moreover, ipo8 zebrafish displayed severe cardiovascular and skeletal defects that mirrored the human phenotype. Our work thus provides evidence that IPO8 plays a critical and non-redundant role in TGF-β signaling during development and reinforces the existing link between TGF-β signaling and connective tissue defects.
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http://dx.doi.org/10.1016/j.ajhg.2021.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206386PMC
June 2021

Intestinal immunoregulation: lessons from human mendelian diseases.

Mucosal Immunol 2021 Apr 15. Epub 2021 Apr 15.

Université de Paris, Imagine Institute, Laboratory of Intestinal Immunity, INSERM UMR 1163, Paris, France.

The mechanisms that maintain intestinal homeostasis despite constant exposure of the gut surface to multiple environmental antigens and to billions of microbes have been scrutinized over the past 20 years with the goals to gain basic knowledge, but also to elucidate the pathogenesis of inflammatory bowel diseases (IBD) and to identify therapeutic targets for these severe diseases. Considerable insight has been obtained from studies based on gene inactivation in mice as well as from genome wide screens for genetic variants predisposing to human IBD. These studies are, however, not sufficient to delineate which pathways play key nonredundant role in the human intestinal barrier and to hierarchize their respective contribution. Here, we intend to illustrate how such insight can be derived from the study of human Mendelian diseases, in which severe intestinal pathology results from single gene defects that impair epithelial and or hematopoietic immune cell functions. We suggest that these diseases offer the unique opportunity to study in depth the pathogenic mechanisms leading to perturbation of intestinal homeostasis in humans. Furthermore, molecular dissection of monogenic intestinal diseases highlights key pathways that might be druggable and therapeutically targeted in common forms of IBD.
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http://dx.doi.org/10.1038/s41385-021-00398-3DOI Listing
April 2021

Loss-of-Function Mutation in PTPN2 Causes Aberrant Activation of JAK Signaling Via STAT and Very Early Onset Intestinal Inflammation.

Gastroenterology 2020 11 25;159(5):1968-1971.e4. Epub 2020 Jul 25.

Université de Paris, Imagine Institute, Laboratory of Intestinal Immunity, Inserm, UMR1163, F-75015, Paris, France. Electronic address:

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http://dx.doi.org/10.1053/j.gastro.2020.07.040DOI Listing
November 2020

Efficacy of Ruxolitinib Therapy in a Patient With Severe Enterocolitis Associated With a STAT3 Gain-of-Function Mutation.

Gastroenterology 2019 03 15;156(4):1206-1210.e1. Epub 2018 Dec 15.

Inserm UMR1163, Intestinal Immunity, Institut Imagine, Paris, France; Université Paris Descartes Sorbonne Paris Cité, Paris, France; AP-HP, Gastroenterology, Hôpital Cochin, Paris, France. Electronic address:

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http://dx.doi.org/10.1053/j.gastro.2018.11.065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433619PMC
March 2019

Circulating biomarkers may be unable to detect infection at the early phase of sepsis in ICU patients: the CAPTAIN prospective multicenter cohort study.

Intensive Care Med 2018 07 30;44(7):1061-1070. Epub 2018 Jun 30.

Service de Médecine Intensive et Réanimation, Groupe Hospitalier Paris Saint-Joseph, Paris, France.

Purpose: Sepsis and non-septic systemic inflammatory response syndrome (SIRS) are the same syndromes, differing by their cause, sepsis being secondary to microbial infection. Microbiological tests are not enough to detect infection early. While more than 50 biomarkers have been proposed to detect infection, none have been repeatedly validated.

Aim: To assess the accuracy of circulating biomarkers to discriminate between sepsis and non-septic SIRS.

Methods: The CAPTAIN study was a prospective observational multicenter cohort of 279 ICU patients with hypo- or hyperthermia and criteria of SIRS, included at the time the attending physician considered antimicrobial therapy. Investigators collected blood at inclusion to measure 29 plasma compounds and ten whole blood RNAs, and-for those patients included within working hours-14 leukocyte surface markers. Patients were classified as having sepsis or non-septic SIRS blindly to the biomarkers results. We used the LASSO method as the technique of multivariate analysis, because of the large number of biomarkers.

Results: During the study period, 363 patients with SIRS were screened, 84 having exclusion criteria. Ninety-one patients were classified as having non-septic SIRS and 188 as having sepsis. Eight biomarkers had an area under the receiver operating curve (ROC-AUC) over 0.6 with a 95% confidence interval over 0.5. LASSO regression identified CRP and HLA-DRA mRNA as being repeatedly associated with sepsis, and no model performed better than CRP alone (ROC-AUC 0.76 [0.68-0.84]).

Conclusions: The circulating biomarkers tested were found to discriminate poorly between sepsis and non-septic SIRS, and no combination performed better than CRP alone.
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http://dx.doi.org/10.1007/s00134-018-5228-3DOI Listing
July 2018

Diagnostic Yield of Next-generation Sequencing in Very Early-onset Inflammatory Bowel Diseases: A Multicentre Study.

J Crohns Colitis 2018 Aug;12(9):1104-1112

GENIUS Group [GENetically ImmUne-mediated enteropathieS] from the European Society for Paediatric Gastroenterology, Hepatology and Nutrition [ESPGHAN].

Background And Aims: An expanding number of monogenic defects have been identified as causative of severe forms of very early-onset inflammatory bowel diseases [VEO-IBD]. The present study aimed at defining how next-generation sequencing [NGS] methods can be used to improve identification of known molecular diagnosis and to adapt treatment.

Methods: A total of 207 children were recruited in 45 paediatric centres through an international collaborative network [ESPGHAN GENIUS working group] with a clinical presentation of severe VEO-IBD [n = 185] or an anamnesis suggestive of a monogenic disorder [n = 22]. Patients were divided at inclusion into three phenotypic subsets: predominantly small bowel inflammation, colitis with perianal lesions, and colitis only. Methods to obtain molecular diagnosis included functional tests followed by specific Sanger sequencing, custom-made targeted NGS, and in selected cases whole exome sequencing [WES] of parents-child trios. Genetic findings were validated clinically and/or functionally.

Results: Molecular diagnosis was achieved in 66/207 children [32%]: 61% with small bowel inflammation, 39% with colitis and perianal lesions, and 18% with colitis only. Targeted NGS pinpointed gene mutations causative of atypical presentations, and identified large exonic copy number variations previously missed by WES.

Conclusions: Our results lead us to propose an optimised diagnostic strategy to identify known monogenic causes of severe IBD.
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http://dx.doi.org/10.1093/ecco-jcc/jjy068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113703PMC
August 2018

Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis.

EMBO Mol Med 2018 04;10(4)

INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France

Herein, we report the first identification of biallelic-inherited mutations in as a Mendelian cause of inflammatory bowel disease in two unrelated patients. encodes for intestinal phosphatase alkaline, a brush border metalloenzyme that hydrolyses phosphate from the lipid A moiety of lipopolysaccharides and thereby drastically reduces Toll-like receptor 4 agonist activity. Prediction tools and structural modelling indicate that all mutations affect critical residues or inter-subunit interactions, and heterologous expression in HEK293T cells demonstrated that all mutations were loss of function. mutations impaired either stability or catalytic activity of ALPI and rendered it unable to detoxify lipopolysaccharide-dependent signalling. Furthermore, ALPI expression was reduced in patients' biopsies, and ALPI activity was undetectable in ALPI-deficient patient's stool. Our findings highlight the crucial role of ALPI in regulating host-microbiota interactions and restraining host inflammatory responses. These results indicate that mutations should be included in screening for monogenic causes of inflammatory bowel diseases and lay the groundwork for ALPI-based treatments in intestinal inflammatory disorders.
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http://dx.doi.org/10.15252/emmm.201708483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5887907PMC
April 2018

First Identification of Biallelic Inherited DUOX2 Inactivating Mutations as a Cause of Very Early Onset Inflammatory Bowel Disease.

Gastroenterology 2017 08 3;153(2):609-611.e3. Epub 2017 Jul 3.

INSERM, UMR1163, Laboratory of Intestinal Immunity and Institut Imagine, Paris, France and GENIUS group from ESPGHAN and Université Paris Descartes-Sorbonne Paris Cité, Paris, France.

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http://dx.doi.org/10.1053/j.gastro.2016.12.053DOI Listing
August 2017

Deficiency in Mucosa-associated Lymphoid Tissue Lymphoma Translocation 1: A Novel Cause of IPEX-Like Syndrome.

J Pediatr Gastroenterol Nutr 2017 03;64(3):378-384

*INSERM, UMR1163, Laboratory of Intestinal Immunity †Université Paris Descartes-Sorbonne Paris Cité and Institut IMAGINE ‡Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Department of Pediatric Gastroenterology, Paris §GENIUS Group (GENetically and/or ImmUne mediated enteropathieS) From ESPGHAN (European Society for Paediatric Gastroenterology, Hepatology and Nutrition) ||Department of Allergology, Rheumatology and Clinical Immunology ¶Department of Haemato-oncology, University Children's Hospital, University Medical Center, Ljubljana, Slovenia #Institut de Minéralogie, de Physique des Matériaux, et de Cosmochimie, Sorbonne Universités-UMR CNRS 7590, UPMC Université Paris 6, Muséum National d'Histoire Naturelle, IRD UMR 206, IUC **Bioinformatics Platform, Université Paris-Descartes-Paris Sorbonne Centre and Institut IMAGINE ††Genomic platform, Institut IMAGINE, Paris, France ‡‡Department of Gastroenterology, Hepatology and Nutrition, University Children's Hospital §§Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia ||||INSERM, UMR1163, Immunogenetics of Pediatric Autoimmunity, Paris, France.

Objective: Early-onset inflammatory bowel diseases can result from a wide spectrum of rare mendelian disorders. Early molecular diagnosis is crucial in defining treatment and in improving life expectancy. Herein we aimed at defining the mechanism of an immunodeficiency-polyendrocrinopathy and enteropathy-X-linked (IPEX)-like disease combined with a severe immunodeficiency in 2 siblings born from distantly related parents.

Methods: Whole exome sequencing was performed on blood-extracted genomic DNA from the 2 affected children and their parents on the genomic platform of Institut IMAGINE. Candidate gene mutation was identified using the in-house software PolyWeb and confirmed by Sanger sequencing. Protein expression was determined by western blot. Flow cytometry was used to assess consequences of the mutation on lymphocyte phenotype and nuclear factor-kappa B (NF-κB) activation at diagnosis and after treatment by hematopoietic stem cell transplantation.

Results: We identified a homozygous missense mutation in mucosa-associated lymphoid tissue lymphoma translocation 1 gene (MALT1), which precluded protein expression. In keeping with the known function of MALT1, NF-κB-dependent lymphocyte activation was severely impaired. Moreover, there was a drastic reduction in Forkhead box P3 (FOXP3) regulatory T cells accounting for the IPEX-like phenotype. Following identification of the mutation, both children received hematopoietic stem cell transplantation, which permitted full clinical recovery. Immunological workup at 6 and 12 months after transplantation showed normal NF-κB activation and correction of regulatory T cells frequency.

Conclusions: Along with FOXP3, interleukin 2 receptor alpha chain (IL2RA), and cytotoxic T-lymphocyte protein 4 precursor (CTLA-4) mutations, MALT1 deficiency should now be considered as a possible cause of IPEX-like syndrome associated with immunodeficiency that can be cured by hematopoietic stem cell transplantation.
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http://dx.doi.org/10.1097/MPG.0000000000001262DOI Listing
March 2017

Reducing hypoxia and inflammation during invasive pulmonary aspergillosis by targeting the Interleukin-1 receptor.

Sci Rep 2016 05 24;6:26490. Epub 2016 May 24.

Unité de recherche Cytokines &Inflammation, Institut Pasteur, Paris.

Hypoxia as a result of pulmonary tissue damage due to unresolved inflammation during invasive pulmonary aspergillosis (IPA) is associated with a poor outcome. Aspergillus fumigatus can exploit the hypoxic microenvironment in the lung, but the inflammatory response required for fungal clearance can become severely disregulated as a result of hypoxia. Since severe inflammation can be detrimental to the host, we investigated whether targeting the interleukin IL-1 pathway could reduce inflammation and tissue hypoxia, improving the outcome of IPA. The interplay between hypoxia and inflammation was investigated by in vivo imaging of hypoxia and measurement of cytokines in the lungs in a model of corticosteroid immunocompromised and in Cxcr2 deficient mice. Severe hypoxia was observed following Aspergillus infection in both models and correlated with development of pulmonary inflammation and expression of hypoxia specific transcripts. Treatment with IL-1 receptor antagonist reduced hypoxia and slightly, but significantly reduced mortality in immunosuppressed mice, but was unable to reduce hypoxia in Cxcr2(-/-) mice. Our data provides evidence that the inflammatory response during invasive pulmonary aspergillosis, and in particular the IL-1 axis, drives the development of hypoxia. Targeting the inflammatory IL-1 response could be used as a potential immunomodulatory therapy to improve the outcome of aspergillosis.
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http://dx.doi.org/10.1038/srep26490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877709PMC
May 2016

Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge.

PLoS One 2016 14;11(4):e0153829. Epub 2016 Apr 14.

Institut Pasteur, Unité Cytokines & Inflammation, Paris, France.

Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153829PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4831689PMC
September 2016

Host response biomarkers in the diagnosis of sepsis: a general overview.

Methods Mol Biol 2015 ;1237:149-211

Unit of Cytokines and Inflammation, Institut Pasteur, 28 rue du Dr Roux, 75724, Paris Cedex 15, France.

Critically ill patients who display a systemic inflammatory response syndrome (SIRS) are prone to develop nosocomial infections. The challenge remains to distinguish as early as possible among SIRS patients those who are developing sepsis. Following a sterile insult, damage-associated molecular patterns (DAMPs) released by damaged tissues and necrotic cells initiate an inflammatory response close to that observed during sepsis. During sepsis, pathogen-associated molecular patterns (PAMPs) trigger the release of host mediators involved in innate immunity and inflammation through identical receptors as DAMPs. In both clinical settings, a compensatory anti-inflammatory response syndrome (CARS) is concomitantly initiated. The exacerbated production of pro- or anti-inflammatory mediators allows their detection in biological fluids and particularly within the bloodstream. Some of these mediators can be used as biomarkers to decipher among the patients those who developed sepsis, and eventually they can be used as prognosis markers. In addition to plasma biomarkers, the analysis of some surface markers on circulating leukocytes or the study of mRNA and miRNA can be helpful. While there is no magic marker, a combination of few biomarkers might offer a high accuracy for diagnosis.
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http://dx.doi.org/10.1007/978-1-4939-1776-1_15DOI Listing
June 2015

Bacterial translocation and plasma cytokines during transcatheter and open-heart aortic valve implantation.

Shock 2015 Jan;43(1):62-7

*Physiology Department, Cochin Academic Hospital, Paris Descartes University, Sorbonne Cite; †Cytokines & Inflammation Unit, Department of Infections and Epidemiology, Pasteur Institute; and ‡Cardiac Surgery Department, Paris Saint-Joseph Hospital, Paris Descartes University, Paris; §Cardiac Surgery Department, Marie-Lannelongue Hospital, Le Plessis-Robinson; and ∥Critical Care Unit, Melun General Hospital, Melun, France.

Objective: To determine whether the good safety profile of transarterial aortic valve implantation (TAVI) is related to lower levels of systemic bacterial translocation and systemic inflammation compared with open-heart surgery.

Background: Transcatheter aortic valve implantation via the transfemoral approach is increasingly used in very high-risk patients with aortic stenosis. The outcomes seem similar to those after open-heart aortic valve replacement (OHAVR).

Methods: Each of 26 consecutive high-risk patients (EuroSCORE >20% for risk of operative death) who underwent TAVI (cases) was matched to the first low-risk patient treated next in our department using elective OHAVR without coronary artery bypass (control subjects). We collected severity, outcome, and echocardiography indicators before and after surgery; complications; proinflammatory cytokine levels; and markers for microbial translocation.

Results: Despite greater illness severity, the TAVI patients had significantly lower vasopressor agent requirements, lower delirium rates, shorter hospital stays, and better hemodynamic findings compared with OHAVR patients. Vascular complications were more common after TAVI than after OHAVR (12, with seven requiring interventional therapy vs. 0, P = 0.006). Patients who underwent TAVI had lower blood transfusion requirements. Two TAVI patients died: one from iliac artery injury and the other from intracardiac prosthesis migration. Patients who underwent TAVI had lower plasma levels of endotoxin and bacterial peptidoglycan, as well as lower proinflammatory cytokine levels, suggesting less gastrointestinal bacterial translocation compared with OHAVR.

Conclusions: Compared with OHAVR, TAVI was associated with decreases in bacterial translocation and inflammation. These differences may explain the lower delirium rate and better hemodynamic stability observed, despite the greater disease severity in TAVI patients.
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http://dx.doi.org/10.1097/SHK.0000000000000262DOI Listing
January 2015

NOD-like receptors in intestinal homeostasis and epithelial tissue repair.

Int J Mol Sci 2014 May 30;15(6):9594-627. Epub 2014 May 30.

Department of Medicine, Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

The intestinal epithelium constitutes a dynamic physical barrier segregating the luminal content from the underlying mucosal tissue. Following injury, the epithelial integrity is restored by rapid migration of intestinal epithelial cells (IECs) across the denuded area in a process known as wound healing. Hence, through a sequence of events involving restitution, proliferation and differentiation of IECs the gap is resealed and homeostasis reestablished. Relapsing damage followed by healing of the inflamed mucosa is a hallmark of several intestinal disorders including inflammatory bowel diseases (IBD). While several regulatory peptides, growth factors and cytokines stimulate restitution of the epithelial layer after injury, recent evidence in the field underscores the contribution of innate immunity in controlling this process. In particular, nucleotide-binding and oligomerization domain-like receptors (NLRs) play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Here, we review the process of intestinal epithelial tissue repair and we specifically focus on the impact of NLR-mediated signaling mechanisms involved in governing epithelial wound healing during disease.
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http://dx.doi.org/10.3390/ijms15069594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100112PMC
May 2014

CD24-triggered caspase-dependent apoptosis via mitochondrial membrane depolarization and reactive oxygen species production of human neutrophils is impaired in sepsis.

J Immunol 2014 Mar 5;192(5):2449-59. Epub 2014 Feb 5.

Unit of Cytokines & Inflammation, Department of Infection and Epidemiology, Institut Pasteur, 75015 Paris, France;

Apoptosis is the most common pathway of neutrophil death under both physiological and inflammatory conditions. In this study, we describe an apoptotic pathway in human neutrophils that is triggered via the surface molecule CD24. In normal neutrophils, CD24 ligation induces death through depolarization of the mitochondrial membrane in a manner dependent on caspase-3 and caspase-9 and reactive oxygen species. Proinflammatory cytokines such as TNF-α, IFN-γ, and GM-CSF upregulated the expression of CD24 in vitro, favoring the emergence of a new CD16(high)/CD24(high) subset of cultured neutrophils. We observed that CD24 expression (at both mRNA and protein levels) was significantly downregulated in neutrophils from sepsis patients but not from patients with systemic inflammatory response syndrome. This downregulation was reproduced by incubation of neutrophils from healthy controls with corticosteroids or with plasma collected from sepsis patients, but not with IL-10 or TGF-β. Decreased CD24 expression observed on sepsis neutrophils was associated with lack of functionality of the molecule, because cross-ligation of CD24 failed to trigger apoptosis in neutrophils from sepsis patients. Our results suggest a novel aspect of CD24-mediated immunoregulation and represent, to our knowledge, the first report showing the role of CD24 in the delayed/defective cell death in sepsis.
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http://dx.doi.org/10.4049/jimmunol.1301055DOI Listing
March 2014

Assessment of efficacy of antifungals against Aspergillus fumigatus: value of real-time bioluminescence imaging.

Antimicrob Agents Chemother 2013 Jul 15;57(7):3046-59. Epub 2013 Apr 15.

Institut Pasteur Unité de Recherche Cytokines & Inflammation, Paris, France.

Aspergillus fumigatus causes life-threatening infections, especially in immunocompromised patients. Common drugs for therapy of aspergillosis are polyenes, azoles, and echinocandins. However, despite in vitro efficacy of these antifungals, treatment failure is frequently observed. In this study, we established bioluminescence imaging to monitor drug efficacy under in vitro and in vivo conditions. In vitro assays confirmed the effectiveness of liposomal amphotericin B, voriconazole, and anidulafungin. Liposomal amphotericin B and voriconazole were fungicidal, whereas anidulafungin allowed initial germination of conidia that stopped elongation but allowed the conidia to remain viable. In vivo studies were performed with a leukopenic murine model. Mice were challenged by intranasal instillation with a bioluminescent reporter strain (5 × 10(5) and 2.5 × 10(5) conidia), and therapy efficacies of liposomal amphotericin B, voriconazole, and anidulafungin were monitored. For monotherapy, the highest treatment efficacy was observed with liposomal amphotericin B, whereas the efficacies of voriconazole and anidulafungin were strongly dependent on the infectious dose. When therapy efficacy was studied with different drug combinations, all combinations improved the rate of treatment success compared to that with monotherapy. One hundred percent survival was obtained for treatment with a combination of liposomal amphotericin B and anidulafungin, which prevented not only pulmonary infections but also infections of the sinus. In conclusion, combination therapy increases treatment success, at least in the murine infection model. In addition, our novel approach based on real-time imaging enables in vivo monitoring of drug efficacy in different organs during therapy of invasive aspergillosis.
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http://dx.doi.org/10.1128/AAC.01660-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697358PMC
July 2013

Interferon-γ and granulocyte/monocyte colony-stimulating factor production by natural killer cells involves different signaling pathways and the adaptor stimulator of interferon genes (STING).

J Biol Chem 2013 Apr 26;288(15):10715-21. Epub 2013 Feb 26.

Institut Pasteur, Unit of Cytokines and Inflammation, Department Infection et Epidémiologie, 28 rue du Dr Roux, F-75015 Paris, France.

Natural killer (NK) cells are important for innate immunity in particular through the production of IFN-γ and GM-CSF. Both cytokines are important in restoration of immune function of tolerized leukocytes under inflammatory events. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors, rarely seeking their protein expression. We previously showed that murine spleen NK cells express TLR9 intracellularly and respond to CpG oligodeoxynucleotide (CpG-ODN) by producing IFN-γ and GM-CSF. However, to get such production the presence of accessory cytokines (such as IL-15 and IL-18) was required, whereas CpG-ODN or accessory cytokines alone did not induce IFN-γ or GM-CSF. We show here that TLR9 overlaps with the Golgi apparatus in NK cells. Furthermore, CpG-ODN stimulation in the presence of accessory cytokines induces the phosphorylation of c-Jun, STAT3, and IκBα. IFN-γ and GM-CSF production requires NF-κB and STAT3 activation as well as Erk-dependent mechanisms for IFN-γ and p38 signaling for GM-CSF. Using knock-out-mice, we show that UNC93b1 and IL-12 (produced by NK cells themselves) are also necessary for IFN-γ and GM-CSF production. IFN-γ production was found to be MyD88- and TLR9-dependent, whereas GM-CSF was TLR9-independent but dependent on STING (stimulator of interferon genes), a cytosolic adaptor recently described for DNA sensing. Our study thereby allows us to gain insight into the mechanisms of synergy between accessory cytokines and CpG-ODN in NK cells. It also identifies a new and alternative signaling pathway for CpG-ODN in murine NK cells.
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http://dx.doi.org/10.1074/jbc.M112.435602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624451PMC
April 2013

Toll-like receptors expression and interferon-γ production by NK cells in human sepsis.

Crit Care 2012 Oct 25;16(5):R206. Epub 2012 Oct 25.

Introduction: During the course of infection, natural killer (NK) cells contribute to innate immunity by producing cytokines, particularly interferon-gamma (IFN-γ). In addition to their beneficial effects against infection, NK cells may play a detrimental role during systemic inflammation, causing lethality during sepsis. Little is known on the immune status of NK cells in patients with systemic inflammatory response syndrome (SIRS) or sepsis in terms of cell surface markers expression and IFN-γ production.

Methods: We investigated 27 sepsis patients and 11 patients with non-infectious SIRS. CD56bright and CD56dim NK cell subsets were identified by flow cytometry and Toll-like receptor (TLR)2, TLR4, TLR9, CX3CR1, CD16 and CD69 expression were analyzed, as well as ex vivo IFN-γ production by NK cells in whole blood samples.

Results: We first showed that in NK cells from healthy controls, TLR2 and TLR4 expression is mainly intracellular, similarly to TLR9. Intracellular levels of TLR2 and TLR4, in both CD56bright and CD56dim NK cell subsets from sepsis patients, were increased compared to healthy subjects. In addition, the percentage of CD69+ cells was higher among NK cells of sepsis patients. No difference was observed for TLR9, CX3CR1, and CD16 expression. The ex vivo stimulation by TLR4 or TLR9 agonists, or whole bacteria in synergy with accessory cytokines (IL-15+IL-18), resulted in significant production of IFN-γ by NK cells of healthy controls. In contrast, for SIRS and sepsis patients this response was dramatically reduced.

Conclusions: This study reports for the first time an intracellular expression of TLR2 and TLR4 in human NK cells. Surface TLR4 expression allows discriminating sepsis and SIRS. Furthermore, during these pathologies, NK cells undergo an alteration of their immune status characterized by a profound reduction of their capacity to release IFN-γ.
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http://dx.doi.org/10.1186/cc11838DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682310PMC
October 2012

DNAemia detection by multiplex PCR and biomarkers for infection in systemic inflammatory response syndrome patients.

PLoS One 2012 15;7(6):e38916. Epub 2012 Jun 15.

Unit Cytokines & Inflammation, Department of Infection and Epidemiology, Institut Pasteur, Paris, France.

Fast and reliable assays to precisely define the nature of the infectious agents causing sepsis are eagerly anticipated. New molecular biology techniques are now available to define the presence of bacterial or fungal DNA within the bloodstream of sepsis patients. We have used a new technique (VYOO®) that allows the enrichment of microbial DNA before a multiplex polymerase chain reaction (PCR) for pathogen detection provided by SIRS-Lab (Jena, Germany). We analyzed 72 sepsis patients and 14 non-infectious systemic inflammatory response syndrome (SIRS) patients. Among the sepsis patients, 20 had a positive blood culture and 35 had a positive microbiology in other biological samples. Of these, 51.4% were positive using the VYOO® test. Among the sepsis patients with a negative microbiology and the non-infectious SIRS, 29.4% and 14.2% were positive with the VYOO® test, respectively. The concordance in bacterial identification between microbiology and the VYOO® test was 46.2%. This study demonstrates that these new technologies offer great hopes, but improvements are still needed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038916PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376137PMC
December 2012

NK cell tolerance to TLR agonists mediated by regulatory T cells after polymicrobial sepsis.

J Immunol 2012 Jun 7;188(12):5850-8. Epub 2012 May 7.

Institut Pasteur, Unité Cytokines et Inflammation, Département Infection et Epidémiologie, F-75015 Paris, France.

As sensors of infection, innate immune cells are able to recognize pathogen-associated molecular patterns by receptors such as TLRs. NK cells present in many tissues contribute to inflammatory processes, particularly through the production of IFN-γ. They may display a protective role during infection but also a detrimental role during sterile or infectious systemic inflammatory response syndrome. Nevertheless, the exact status of NK cells during bacterial sepsis and their capacity directly to respond to TLR agonists remain unclear. The expression of TLRs in NK cells has been widely studied by analyzing the mRNA of these receptors. The aim of this study was to gain insight into TLR2/TLR4/TLR9 expression on/in murine NK cells at the protein level and determine if their agonists were able to induce cytokine production. We show, by flow cytometry, a strong intracellular expression of TLR2 and a low of TLR4 in freshly isolated murine spleen NK cells, similar to that of TLR9. In vitro, purified NK cells respond to TLR2, TLR4, and TLR9 agonists, in synergy with activating cytokines (IL-2, IL-15, and/or IL-18), and produce proinflammatory cytokines (IFN-γ and GM-CSF). Finally, we explored the possible tolerance of NK cells to TLR agonists after a polymicrobial sepsis (experimental peritonitis). For the first time, to our knowledge, NK cells are shown to become tolerant in terms of proinflammatory cytokines production after sepsis. We show that this tolerance is associated with a reduction of the CD27(+)CD11b(-) subset in the spleen related to the presence of regulatory T cells and mainly mediated by TGF-β.
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http://dx.doi.org/10.4049/jimmunol.1103616DOI Listing
June 2012

Mechanisms of TNF induction by heat-killed Staphylococcus aureus differ upon the origin of mononuclear phagocytes.

Am J Physiol Cell Physiol 2011 Apr 5;300(4):C850-9. Epub 2011 Jan 5.

Institut Pasteur, Cytokines & Inflammation Unit, Paris, France.

Mononuclear phagocytes are among the first immune cells activated after pathogens invasion. Although they all derive from the same progenitor in the bone marrow, their characteristics differ on the compartment from which they are derived. In this work, we investigated the contribution of phagocytosis for tumor necrosis factor (TNF) production by murine mononuclear phagocytes (monocytes, peritoneal and alveolar macrophages) in response to heat-killed Staphylococcus aureus (HKSA). Mononuclear phagocytes behaved differently, depending on their compartment of residence. Indeed, when bacterial uptake or phagosome maturation was blocked, activation through membrane receptors was sufficient for a maximal production of TNF and interleukin-10 by peritoneal macrophages. In contrast, monocytes, and to a lesser extent alveolar macrophages, required phagocytosis for optimal cytokine production. While investigating the different actors of signalization, we found that p38 kinase and phosphatidylinositol 3-kinase were playing an important role in HKSA phagocytosis and TNF production. Furthermore, blocking the α(5)β(1)-integrin significantly decreased TNF production in response to HKSA in all three cell types. Finally, using mononuclear phagocytes from NOD2 knockout mice, we observed that TNF production in response to HKSA was dependent on NOD2 for monocytes and peritoneal macrophages. In conclusion, we demonstrate that the mechanisms of activation leading to TNF production in response to HKSA are specific for each mononuclear phagocyte population and involve different recognition processes and signaling pathways. The influence of the compartments on cell properties and behavior should be taken into account, to better understand cell physiology and host-pathogen interaction, and to define efficient strategies to fight infection.
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http://dx.doi.org/10.1152/ajpcell.00187.2010DOI Listing
April 2011

Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice.

PLoS One 2010 Jul 22;5(7):e11720. Epub 2010 Jul 22.

Faculty of Biotechnology, University of Naples, Portici, Naples, Italy.

In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the M(Sa) phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-alpha, IFN-gamma and Il-1beta genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 microl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 microg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011720PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908692PMC
July 2010

Contribution of NOD2 to lung inflammation during Staphylococcus aureus-induced pneumonia.

Microbes Infect 2010 Sep 21;12(10):759-67. Epub 2010 May 21.

Institut Pasteur, Unité Cytokines & Inflammation, Département Infection et épidémiologie, 28 rue du Dr Roux, F-75015 Paris, France.

Staphylococcus aureus is the most commonly found Gram-positive bacterium in patients admitted in intensive-care units, causing septicaemia or pneumonia. In this work, we investigated the role of NOD2 in S. aureus-induced pneumonia. We found that the absence of NOD2 affected weight loss and recovery speed. Nod2-/- mice showed a reduced lung inflammation in comparison to wild-type animals, with lower presence of cytokines in broncho-alveolar lavage fluids and reduced recruitment of neutrophils. Furthermore, histological analysis of the lungs revealed less severe lesions in Nod2-/- mice at day 2 and day 7 post-infection. In conclusion, we demonstrated that NOD2 is not a crucial receptor to fight S. aureus-induced pneumonia, but that it contributes to the inflammatory response in the lungs. Interestingly, the absence of NOD2 led to a lesser inflammation and was finally beneficial for the animal recovery.
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http://dx.doi.org/10.1016/j.micinf.2010.05.003DOI Listing
September 2010

Bacteriophage therapy of Salmonella enterica: a fresh appraisal of bacteriophage therapy.

J Infect Dis 2010 Jan;201(1):52-61

Faculty of Biotechnology, University of Naples Federico II, Naples, Italy.

Background: The most serious criticisms leveled at bacteriophage therapy are as follows: phages induce neutralizing antibodies, phages are active only when administered shortly after bacterial infection, and phage-resistant bacteria emerge rapidly in the course of therapy.

Methods: Phages lytic for several Salmonella enterica serovars were isolated by means of standard protocols from feces of patients with gastroenteritis. Growth of S. enterica serovar Paratyphi B (Salp572(phi1S)) in the presence of phage phi1 (selected from among 8 phages for its larger host range) provided a phage phi1-resistant bacterial strain (Salp572(phi1R)). The properties of the Salp572(phi1S) and Salp572(phi1R) strains and of phage phi1 were studied in a mouse model of experimental infection.

Results: Phages induced nonneutralizing antibodies and were active 2 weeks after experimental infection of mice; phage-resistant bacteria were avirulent and short lived in vivo. More importantly, phage-resistant bacteria were excellent vaccines, protecting against lethal doses of heterologous S. enterica serovars.

Conclusions: Phage therapy effectiveness has not yet been properly assessed.
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http://dx.doi.org/10.1086/648478DOI Listing
January 2010

Experimental phage therapy against Staphylococcus aureus in mice.

Antimicrob Agents Chemother 2007 Aug 21;51(8):2765-73. Epub 2007 May 21.

Dipartimento di Scienze del Suolo, della Pianta, dell'Ambiente e delle Produzioni Animali, Università degli Studi di Napoli Federico II, Via Università 133, Portici, Naples, Italy.

The present study describes a bacteriophage (M(Sa)) active against Staphylococcus aureus, including methicillin-resistant staphylococcal strains. When inoculated into mice simultaneously with S. aureus A170 (10(8) CFU/mouse), phage (10(9) PFU) rescued 97% of the mice; when applied to nonlethal (5 x 10(6) CFU/mouse) 10-day infections, the phage also fully cleared the bacteria. The phage M(Sa), delivered inside macrophages by S. aureus, kills the intracellular staphylococci in vivo and in vitro. The phage can also prevent abscess formation and reduce the bacterial load and weight of abscesses. These results suggest a potential use of the phage for the control of both local and systemic human S. aureus infections.
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http://dx.doi.org/10.1128/AAC.01513-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932491PMC
August 2007

Small surface-associated factors mediate adhesion of a food-isolated strain of Lactobacillus fermentum to Caco-2 cells.

Res Microbiol 2005 Aug;156(7):830-6

Dipartimento di Biologia Strutturale e Funzionale, Università Federico II, Napoli, Italy.

In a search for bacteria having putative probiotic activity, we screened a collection of food-isolated microorganisms for the ability to survive at low pH in the presence of bile salts and for the production of antimicrobial compounds active against a number of animal pathogens. Among these, we found a strain that we classified as a member of Lactobacillus fermentum sp., and we further investigated its features. This organism was able to adhere to human enterocyte-like (Caco-2) cells with high efficiency as compared to that of a well known indicator strain. Chromatographic analysis indicated that at least two small (less than 3 kDa) factors were involved in mediating the in vitro interaction of L. fermentum with Caco-2 cells. Adhesion activity could be abolished by mild treatment of the bacterial cells in buffer and rescued by incubating them with either the same buffer after its use in the treatment or with chromatographic fractions containing each of the two factors, which indicated that these factors were loosely associated with the cell wall and that each of them was sufficient to warrant the adhesiveness of L. fermentum to Caco-2 cells. These data are suggestive of a novel mechanism of bacterial adhesion to epithelial cells.
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http://dx.doi.org/10.1016/j.resmic.2005.05.001DOI Listing
August 2005
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