Publications by authors named "Emma Lefrançais"

14 Publications

  • Page 1 of 1

Cystic fibrosis transmembrane conductance regulator dysfunction in platelets drives lung hyperinflammation.

J Clin Invest 2020 04;130(4):2041-2053

Department of Medicine, UCSF, San Francisco, California, USA.

Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in CF, and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets and TRPC6 are what we believe to be novel therapeutic targets in the treatment of CF lung disease.
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http://dx.doi.org/10.1172/JCI129635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108932PMC
April 2020

Mitochondrial DNA Stimulates TLR9-Dependent Neutrophil Extracellular Trap Formation in Primary Graft Dysfunction.

Am J Respir Cell Mol Biol 2020 03;62(3):364-372

Department of Medicine.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.
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http://dx.doi.org/10.1165/rcmb.2019-0140OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055700PMC
March 2020

Platelet Biogenesis in the Lung Circulation.

Physiology (Bethesda) 2019 11;34(6):392-401

Departments of Medicine and Laboratory Medicine, University of California, San Francisco, CA.

Megakaryocytes are normal cellular components of the blood returning to the heart and entering the lungs, and historical data has pointed to a role of the lungs in platelet production. Recent studies using intravital microscopy have demonstrated that platelet release occurs in the lung from bone marrow megakaryocytes that embolize into the lung circulation.
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http://dx.doi.org/10.1152/physiol.00017.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957358PMC
November 2019

Extracellular DNA, Neutrophil Extracellular Traps, and Inflammasome Activation in Severe Asthma.

Am J Respir Crit Care Med 2019 05;199(9):1076-1085

1 Division of Pulmonary and Critical Care Medicine, Department of Medicine, and.

Extracellular DNA (eDNA) and neutrophil extracellular traps (NETs) are implicated in multiple inflammatory diseases. NETs mediate inflammasome activation and IL-1β secretion from monocytes and cause airway epithelial cell injury, but the role of eDNA, NETs, and IL-1β in asthma is uncertain. To characterize the role of activated neutrophils in severe asthma through measurement of NETs and inflammasome activation. We measured sputum eDNA in induced sputum from 399 patients with asthma in the Severe Asthma Research Program-3 and in 94 healthy control subjects. We subdivided subjects with asthma into eDNA-low and -high subgroups to compare outcomes of asthma severity and of neutrophil and inflammasome activation. We also examined if NETs cause airway epithelial cell damage that can be prevented by DNase. We found that 13% of the Severe Asthma Research Program-3 cohort is "eDNA-high," as defined by sputum eDNA concentrations above the upper 95th percentile value in health. Compared with eDNA-low patients with asthma, eDNA-high patients had lower Asthma Control Test scores, frequent history of chronic mucus hypersecretion, and frequent use of oral corticosteroids for maintenance of asthma control (all values <0.05). Sputum eDNA in asthma was associated with airway neutrophilic inflammation, increases in soluble NET components, and increases in caspase 1 activity and IL-1β (all values <0.001). In studies, NETs caused cytotoxicity in airway epithelial cells that was prevented by disruption of NETs with DNase. High extracellular DNA concentrations in sputum mark a subset of patients with more severe asthma who have NETs and markers of inflammasome activation in their airways.
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http://dx.doi.org/10.1164/rccm.201810-1869OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6515873PMC
May 2019

LPS-induced Lung Platelet Recruitment Occurs Independently from Neutrophils, PSGL-1, and P-Selectin.

Am J Respir Cell Mol Biol 2019 08;61(2):232-243

1Sackler Institute of Pulmonary Pharmacology, Institute of Pharmaceutical Science and.

Platelets are recruited to inflammatory foci and contribute to host defense and inflammatory responses. Compared with platelet recruitment in hemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defense are poorly understood. Neutrophil recruitment to lung airspaces after inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice after LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils. BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and, 48 hours later, lungs were collected and platelets and neutrophils quantified in tissue sections by immunohistochemistry. The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules, P-selectin or PSGL-1, or treatment with a neutrophil-depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre × mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs. Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation. In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.
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http://dx.doi.org/10.1165/rcmb.2018-0182OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6670039PMC
August 2019

Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury.

JCI Insight 2018 02 8;3(3). Epub 2018 Feb 8.

Department of Medicine and.

Neutrophils dominate the early immune response in pathogen-induced acute lung injury, but efforts to harness their responses have not led to therapeutic advancements. Neutrophil extracellular traps (NETs) have been proposed as an innate defense mechanism responsible for pathogen clearance, but there are concerns that NETs may induce collateral damage to host tissues. Here, we detected NETs in abundance in mouse models of severe bacterial pneumonia/acute lung injury and in human subjects with acute respiratory distress syndrome (ARDS) from pneumonia or sepsis. Decreasing NETs reduced lung injury and improved survival after DNase I treatment or with partial protein arginine deiminase 4 deficiency (PAD4+/-). Complete PAD4 deficiency (PAD4-/-) reduced NETs and lung injury but was counterbalanced by increased bacterial load and inflammation. Importantly, we discovered that the lipoxin pathway could be a potent modulator of NET formation, and that mice deficient in the lipoxin receptor (Fpr2-/-) produced excess NETs leading to increased lung injury and mortality. Lastly, we observed in humans that increased plasma NETs were associated with ARDS severity and mortality, and lower plasma DNase I levels were associated with the development of sepsis-induced ARDS. We conclude that a critical balance of NETs is necessary to prevent lung injury and to maintain microbial control, which has important therapeutic implications.
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http://dx.doi.org/10.1172/jci.insight.98178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821185PMC
February 2018

[The hidden face of the lung: a platelet factory and a blood progenitors reservoir].

Med Sci (Paris) 2017 Dec 20;33(12):1032-1035. Epub 2017 Dec 20.

Université de Californie, San Francisco HSE 1355A - 513 parnassus ave, San Francisco, CA-94143-0130, États-Unis.

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http://dx.doi.org/10.1051/medsci/20173312005DOI Listing
December 2017

Neutralizing Extracellular Histones in Acute Respiratory Distress Syndrome. A New Role for an Endogenous Pathway.

Am J Respir Crit Care Med 2017 07;196(2):122-124

1 Department of Medicine University of California, San Francisco San Francisco, California.

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http://dx.doi.org/10.1164/rccm.201701-0095EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955066PMC
July 2017

The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.

Nature 2017 04 22;544(7648):105-109. Epub 2017 Mar 22.

Department of Medicine, University of California, San Francisco (UCSF), San Francisco, California 94143, USA.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.
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http://dx.doi.org/10.1038/nature21706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663284PMC
April 2017

Central domain of IL-33 is cleaved by mast cell proteases for potent activation of group-2 innate lymphoid cells.

Proc Natl Acad Sci U S A 2014 Oct 13;111(43):15502-7. Epub 2014 Oct 13.

Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France; Université de Toulouse, Université Paul Sabatier, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France; and

Interleukin-33 (IL-33) is an alarmin cytokine from the IL-1 family. IL-33 activates many immune cell types expressing the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2, including group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes), the major producers of IL-5 and IL-13 during type-2 innate immune responses and allergic airway inflammation. IL-33 is likely to play a critical role in asthma because the IL33 and ST2/IL1RL1 genes have been reproducibly identified as major susceptibility loci in large-scale genome-wide association studies. A better understanding of the mechanisms regulating IL-33 activity is thus urgently needed. Here, we investigated the role of mast cells, critical effector cells in allergic disorders, known to interact with ILC2s in vivo. We found that serine proteases secreted by activated mast cells (chymase and tryptase) generate mature forms of IL-33 with potent activity on ILC2s. The major forms produced by mast cell proteases, IL-33(95-270), IL-33(107-270), and IL-33(109-270), were 30-fold more potent than full-length human IL-33(1-270) for activation of ILC2s ex vivo. They induced a strong expansion of ILC2s and eosinophils in vivo, associated with elevated concentrations of IL-5 and IL-13. Murine IL-33 is also cleaved by mast cell tryptase, and a tryptase inhibitor reduced IL-33-dependent allergic airway inflammation in vivo. Our study identifies the central cleavage/activation domain of IL-33 (amino acids 66-111) as an important functional domain of the protein and suggests that interference with IL-33 cleavage and activation by mast cell and other inflammatory proteases could be useful to reduce IL-33-mediated responses in allergic asthma and other inflammatory diseases.
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http://dx.doi.org/10.1073/pnas.1410700111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4217470PMC
October 2014

Mechanisms of IL-33 processing and secretion: differences and similarities between IL-1 family members.

Eur Cytokine Netw 2012 Oct-Dec;23(4):120-7

Centre national de la recherche scientifique, Institut de pharmacologie et de biologie structurale, 205 route de Narbonne F-31077 Toulouse, France, Université de Toulouse, universite Paul Sabatier, institut de pharmacologie et de biologie structurale, F-31077 Toulouse, France.

Interleukin-33 (IL-33) is the latest member of the IL-1 family that has become very attractive since the discovery of its major target cells, the innate lymphoid cells type 2 (ILC2), involved in the initiation of the type 2 immune response (secretion of IL-5 and IL-13) during parasitic infection and allergic diseases such as asthma. IL-33 is a chromatin-associated protein as it possesses in its N-terminus, a chromatin-binding domain, and is constitutively expressed in the nuclei of endothelial cells and in epithelial cells of tissues exposed to the environment. It is however, essential for IL-33 to be extracellularly released to bind to its receptor ST2 through the C-terminus portion of the protein in order to induce the inflammatory and type 2 responses. Like other IL-1 family members, IL-33 does not possess any signal peptide and may be released through unconventional secretory mechanisms or following cell damage and necrosis. It was initially believed that IL-33, like IL-1β and IL-18, requires processing by caspase-1 to be released, and for biological activity. On the contrary, full length IL-33 is biologically active, and processing by caspases results rather in IL-33 inactivation. Moreover, it has been recently shown that the bioactivity of IL-33 can be increased by inflammatory proteases secreted in the microenvironment, similarly to IL-1α, IL-1β and IL-18. This review will summarize recent progress on how IL-33 is released and processed compared with the other IL-1 family members, and how the immune cells recruited to the site of injury can regulate the disease-associated function of IL-33.
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http://dx.doi.org/10.1684/ecn.2012.0320DOI Listing
July 2013

Neutralisation of the interleukin-33/ST2 pathway ameliorates experimental colitis through enhancement of mucosal healing in mice.

Gut 2013 Dec 21;62(12):1714-23. Epub 2012 Nov 21.

CNRS and University, UMR7355, Molecular Immunology, Orleans, France and Institute of Infectious Disease and Molecular Medicine, University of Cape Town, RSA.

Objective: Inflammatory bowel diseases (IBD) have been intrinsically linked to a deregulated cytokine network, but novel therapeutic principles are urgently needed. Here we identify the interleukin (IL)-33 and its receptor ST2 as key negative regulators of wound healing and permeability in the colon of mice.

Design: Expression of IL-33 and ST2 was determined by qRT-PCR, ELISA, immunohistochemistry and western-blot analysis. Wild-type and St2(-/-) mice were used in wound healing experiments and in two experimental models of IBD triggered by 2,4,6-trinitrobenzene sulphonic acid or dextran sodium sulphate (DSS). Neutralisation of ST2 was performed by using a specific blocking antibody.

Results: Nuclear localisation and enhanced expression of IL-33 in myofibroblasts and enterocytes was linked to disease involvement independently of inflammation, while the expression of ST2 was primarily restricted to the colonic epithelia. In two experimental models of IBD, genetic ablation of ST2 significantly improved signs of colitis, while a sustained epithelial expression of the cyto-protective factor connexin-43 was observed in DSS-treated St2-deficient mice. Unexpectedly, absence of ST2 in non-hematopoietic cells was sufficient to protect against colitis. Consistently, specific inhibition of endogenous ST2-mediated signalling by treatment with neutralising antibody improved DSS-induced colitis. In addition, IL-33 treatment impaired epithelial barrier permeability in vitro and in vivo, whereas absence of ST2 enhanced wound healing response upon acute mechanical injury in the colon.

Conclusions: Our study unveiled a novel non-hematopoietic function of IL-33 in epithelial barrier function and wound healing. Therefore, blocking the IL-33/ST2 axis may represent an efficient therapy in IBD.
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http://dx.doi.org/10.1136/gutjnl-2011-301785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841767PMC
December 2013

Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.

J Immunol 2012 Apr 27;188(7):3488-95. Epub 2012 Feb 27.

Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France.

IL-33 (previously known as NF from high endothelial venules) is an IL-1 family cytokine that signals through the ST2 receptor and drives cytokine production in mast cells, basophils, eosinophils, invariant NKT and NK cells, Th2 lymphocytes, and type 2 innate immune cells (natural helper cells, nuocytes, and innate helper 2 cells). Little is known about endogenous IL-33; for instance, the cellular sources of IL-33 in mouse tissues have not yet been defined. In this study, we generated an Il-33-LacZ gene trap reporter strain (Il-33(Gt/Gt)) and used this novel tool to analyze expression of endogenous IL-33 in vivo. We found that the Il-33 promoter exhibits constitutive activity in mouse lymphoid organs, epithelial barrier tissues, brain, and embryos. Immunostaining with anti-IL-33 Abs, using Il-33(Gt/Gt) (Il-33-deficient) mice as control, revealed that endogenous IL-33 protein is highly expressed in mouse epithelial barrier tissues, including stratified squamous epithelia from vagina and skin, as well as cuboidal epithelium from lung, stomach, and salivary gland. Constitutive expression of IL-33 was not detected in blood vessels, revealing the existence of species-specific differences between humans and mice. Importantly, IL-33 protein was always localized in the nucleus of producing cells with no evidence for cytoplasmic localization. Finally, strong expression of the Il-33-LacZ reporter was also observed in inflamed tissues, in the liver during LPS-induced endotoxin shock, and in the lung alveoli during papain-induced allergic airway inflammation. Together, our findings support the possibility that IL-33 may function as a nuclear alarmin to alert the innate immune system after injury or infection in epithelial barrier tissues.
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http://dx.doi.org/10.4049/jimmunol.1101977DOI Listing
April 2012

IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G.

Proc Natl Acad Sci U S A 2012 Jan 17;109(5):1673-8. Epub 2012 Jan 17.

Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, F-31077 Toulouse, France.

Interleukin-33 (IL-33) (NF-HEV) is a chromatin-associated nuclear cytokine from the IL-1 family, which has been linked to important diseases, including asthma, rheumatoid arthritis, ulcerative colitis, and cardiovascular diseases. IL-33 signals through the ST2 receptor and drives cytokine production in type 2 innate lymphoid cells (ILCs) (natural helper cells, nuocytes), T-helper (Th)2 lymphocytes, mast cells, basophils, eosinophils, invariant natural killer T (iNKT), and natural killer (NK) cells. We and others recently reported that, unlike IL-1β and IL-18, full-length IL-33 is biologically active independently of caspase-1 cleavage and that processing by caspases results in IL-33 inactivation. We suggested that IL-33, which is released upon cellular damage, may function as an endogenous danger signal or alarmin, similar to IL-1α or high-mobility group box 1 protein (HMGB1). Here, we investigated the possibility that IL-33 activity may be regulated by proteases released during inflammation. Using a combination of in vitro and in vivo approaches, we demonstrate that neutrophil serine proteases cathepsin G and elastase can cleave full-length human IL-33(1-270) and generate mature forms IL-33(95-270), IL-33(99-270), and IL-33(109-270). These forms are produced by activated human neutrophils ex vivo, are biologically active in vivo, and have a ~10-fold higher activity than full-length IL-33 in cellular assays. Murine IL-33 is also cleaved by neutrophil cathepsin G and elastase, and both full-length and cleaved endogenous IL-33 could be detected in the bronchoalveolar lavage fluid in an in vivo model of acute lung injury associated with neutrophil infiltration. We propose that the inflammatory microenvironment may exacerbate disease-associated functions of IL-33 through the generation of highly active mature forms.
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http://dx.doi.org/10.1073/pnas.1115884109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277172PMC
January 2012