66 results on '"C. Benarafa"'
Search Results
2. Role of the chemokine eotaxin in the pathogenesis of equine sweet itch
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C, Benarafa, M E, Collins, A S, Hamblin, and F M, Cunningham
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Chemokine CCL11 ,Biopsy ,Pruritus ,Receptors, CCR3 ,Insect Bites and Stings ,Ceratopogonidae ,Monocyte Chemoattractant Proteins ,Up-Regulation ,Eosinophils ,Case-Control Studies ,Chemokines, CC ,Dermatitis, Allergic Contact ,Animals ,Horse Diseases ,Receptors, Chemokine ,Horses ,RNA, Messenger ,Saliva ,Chemokine CCL2 ,Skin - Abstract
The chemokine eotaxin is involved in the recruitment of eosinophils and T helper 2 lymphocytes in human allergic diseases, and drugs that block its activity, including eotaxin receptor (CCR3) antagonists, are being developed. The authors have recently cloned the horse ortholog of eotaxin and shown that it can induce equine eosinophil migration and activation in vitro. Moreover, eotaxin mRNA expression was upregulated in cultured horse dermal fibroblasts exposed to equine interleukin-4, suggesting a possible source of this eosinophil chemoattractant in equine skin. The results of this study show that eotaxin and monocyte chemoattractant protein (MCP) 1, but not MCP-2 or MCP-4, mRNA expression is upregulated in skin biopsies of sweet itch lesions when eosinophils are present, when compared with clinically normal skin from the same ponies. more...
- Published
- 2002
Catalog
3. Madin Darby bovine kidney cell synchronization by lovastatin: application to bovine herpesvirus-1 gene expression
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A, Vanderplasschen, E, Hanon, C, Benarafa, R, Greimers, A, Op De Beeck, M, Loncar, and P P, Pastoret
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Gene Expression Regulation, Viral ,Time Factors ,Cell Cycle ,Animals ,Cattle ,Lovastatin ,Flow Cytometry ,Kidney ,Cell Line ,Herpesvirus 1, Bovine - Abstract
The number of investigations involving cell proliferation has increased rapidly in the last years. One of the major difficulties in studying cell-cycle-related events is obtaining highly synchronous cell populations without metabolic imbalance. This study demonstrates that the Madin Darby bovine kidney (MDBK) cells, a commonly used cell line in veterinary research, can be effectively synchronized using lovastatin (Lov), a drug used to treat hypercholesteremia in humans. This was demonstrated by the following results: (i) Lov inhibits cell proliferation in a dose-dependent manner; (ii) Lov synchronizes MDBK cells mainly in the G1 and secondarily in the G2+M cell-cycle phases; (iii) the cytostatic effect of Lov can be specifically inhibited by addition of mevalonate (Mev) (Lov inhibits the synthesis of Mev); (iv) removal of Lov from G1-arrested cultures, followed by addition of Mev, resulted in the synchronous recovery of DNA synthesis; and (v) 5-bromo2'-deoxyuridine incorporation experiments revealed that MDBK cells synchronization by Lov can be followed for at least 3 cycles after removal of Lov and addition of Mev. Furthermore, as an application of investigations based on the availability of synchronized MDBK, we showed that bovine herpesvirus-1 gene expression is independent on the cell cycle. more...
- Published
- 1994
4. SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .
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Grin PM, Baid K, de Jesus HCR, Kozarac N, Bell PA, Jiang SZ, Kappelhoff R, Butler GS, Leborgne NGF, Pan C, Pablos I, Machado Y, Vederas JC, Kim H, Benarafa C, Banerjee A, and Overall CM
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- Animals, Humans, Mice, Caspases metabolism, Intracellular Signaling Peptides and Proteins metabolism, Mice, Inbred C57BL, Pore Forming Cytotoxic Proteins metabolism, Mice, Knockout, HEK293 Cells, Neoplasm Proteins metabolism, Enzyme Activation, Apoptosis Regulatory Proteins metabolism, Gasdermins, SARS-CoV-2 metabolism, Phosphate-Binding Proteins metabolism, Pyroptosis, Coronavirus 3C Proteases metabolism, COVID-19 virology, COVID-19 metabolism, COVID-19 pathology
- Abstract
SARS-CoV-2 3C-like protease (3CL
pro or Mpro ) cleaves the SARS-CoV-2 polyprotein and >300 intracellular host proteins to enhance viral replication. By lytic cell death following gasdermin (GSDM) pore formation in cell membranes, antiviral pyroptosis decreases 3CLpro expression and viral replication. Unexpectedly, 3CLpro and nucleocapsid proteins undergo unconventional secretion from infected cells via caspase-activated GSDMD/E pores in the absence of cell lysis. Bronchoalveolar lavage fluid of wild-type SARS-CoV-2-infected mice contains 3CLpro , which decreases in Gsdmd-/- Gsdme-/- mice. We identify new 3CLpro cut-sites in GSDMD at LQ29 ↓30 SS, which blocks pore formation by 3CLpro cleavage at LH270 ↓N lying adjacent to the caspase activation site (NFLTD275 ↓G). Cleavage inactivation of GSDMD prevents excessive pore formation, thus countering antiviral pyroptosis and increasing 3CLpro secretion. Extracellular 3CLpro retains activity in serum, dampens platelet activation and aggregation, and inactivates antiviral interferon-λ1. Thus, in countering gasdermin pore formation and pyroptosis in SARS-CoV-2 infection, 3CLpro is secreted with extracellular pathological sequelae., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.) more...- Published
- 2024
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5. Cigarette smoke-induced disordered microbiota aggravates the severity of influenza A virus infection.
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Wüthrich T, de Brot S, Richina V, Mostacci N, Baumann Z, Leborgne NGF, Godel A, Alves MP, Bentires-Alj M, Benarafa C, and Hilty M
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- Animals, Mice, Influenza A Virus, H1N1 Subtype, Female, Influenza A virus, Feces microbiology, Severity of Illness Index, Gastrointestinal Microbiome, Male, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections microbiology, Mice, Inbred C57BL, Microbiota
- Abstract
Cigarette smoke (CS) promotes the development of chronic pulmonary disease and has been associated with increased risk for influenza-related illness. Here, we directly addressed the impact of CS disordered microbiota on the severity of influenza A virus (IAV) infection. Specific and opportunistic pathogen-free (SOPF) C57BL/6J mice were exposed to CS or room air (RA) for 5.5 months. Each exposed mouse was then cohoused with a group of recipient germ-free (GF) mice for 1 month for microbial transfer. Colonized GF mice were then infected intranasally with IAV and disease development was monitored. Upper and lower airway and fecal microbiota were longitudinally investigated by 16S rRNA gene sequencing and bacterial cultures in donor and recipient mice. The bacterial family Streptococcaceae accounted for the largest difference between CS- and RA-exposed microbiota in the oropharynx. Analysis of the oropharynx and fecal microbiota indicated an efficient transfer to coprophagic recipient mice, which replicated the differences in microbiota composition observed in donor mice. Subsequent IAV infection revealed significantly higher weight loss for CS microbiota recipient mice at 8-10 days post infection (dpi) compared to control recipient mice. In addition, H1N1 infection inflicted substantial changes in the microbiota composition, especially at days 4 and 8 after infection. In conclusion, mice with a CS-associated microbiota suffer from higher disease severity upon IAV infection compared to mice colonized with a normal SOPF microbiota. Our data suggest that independently of CS exposure and concomitant structural lung damage, microbial distortion due to CS exposure may impact the severity of IAV disease course.IMPORTANCEIt has been reported that chronic exposure to CS is associated with a disordered microbiota composition. In this study, we colonized germ-free (GF) mice with the microbiota from SOPF mice which were chronically exposed to CS or RA. This allowed disentangling the effect of the disordered microbiota from the immune-modulating effects of actual CS exposure. We observed a successful transfer of the microbiotas after cohousing including specific microbiota differences induced by CS exposure in formerly GF mice, which were never exposed to CS. We then investigated the effects of IAV infection on the disease course and microbiotas of formerly GF mice. We found that mice with CS-associated microbiota reveal worse disease course compared to the control group. We hypothesize that CS-induced disordering of the microbiota may, indeed, impact the severity of influenza A disease., Competing Interests: The authors declare no conflict of interest. more...
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- 2024
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6. Mosquito-independent milk-associated transmission of zoonotic Wesselsbron virus in sheep.
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Zimoch M, Grau-Roma L, Liniger M, Donzé N, Godel A, Escribano D, Trüeb BS, Pramateftaki P, Torres-Puig S, Cerón JJ, Thiel V, Jores J, Summerfield A, Ruggli N, Benarafa C, and García-Nicolás O
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- Animals, Sheep, Female, Milk virology, Aedes virology, Zoonoses transmission, Zoonoses virology, Flavivirus pathogenicity, Mosquito Vectors virology, Viral Zoonoses transmission, Viral Zoonoses virology, Sheep Diseases virology, Sheep Diseases transmission
- Abstract
Wesselsbron virus (WSLV) is a zoonotic, mosquito-borne orthoflavivirus endemic to sub-Saharan Africa, causing abortions and stillbirths in small ruminants. The life cycle of WSLV involves Aedes mosquitoes and various wildlife and domestic animals. Seminal studies in the 1950s have shown the zoonotic potential of WSLV, notably in accidental infections of laboratory workers exposed to infected material. More recent epidemiological studies suggest the emergence of clade I WSLV strains in peri-domestic and rural areas of western and eastern Africa. The pathobiology of recent clade I WSLV strains is unknown and no virus isolate is available. To address these gaps, we generated a recombinant clade I WSLV SA999 infectious clone (rSA999) by reverse genetics. Subsequently, lactating ewes were inoculated intravenously with the WSLV rSA999 strain or the clade II SAH177 strain in insect-free biocontainment stables. Inoculated ewes developed fever, viremia, and showed high levels of viral RNA at mucosal surfaces, and elevated viral titers in milk. Milk production was reduced, which directly affected the growth of the lambs, particularly within the rSA999 group. The ewes with higher WSLV titers in their milk in each group transmitted the infection to their lambs, which developed fever, prolonged viremia, and virus secretion. All infected animals produced high antibody titers with cross-neutralizing activity against both WSLV strains. Histopathology and blood biochemistry analysis indicated liver damage associated with necrotizing hepatitis lesions and active viral replication in some cases, which was more pronounced in the rSA999 group. Notably, only the SAH177-infected animals exhibited lesions consistent with meningoencephalitis, suggesting that WSLV clade II strains are neurotropic and that clade I strain are more hepatotropic. These findings demonstrate a previously unrecognized mode of vector-free transmission of WSLV that raises significant concerns for public and animal health., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zimoch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.) more...
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- 2024
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7. A safe, effective and adaptable live-attenuated SARS-CoV-2 vaccine to reduce disease and transmission using one-to-stop genome modifications.
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Schön J, Barut GT, Trüeb BS, Halwe NJ, Berenguer Veiga I, Kratzel A, Ulrich L, Kelly JN, Brügger M, Wylezich C, Taddeo A, Aguiar Moreira E, Túrós D, Grau-Roma L, Ahrens AK, Schlottau K, Britzke T, Breithaupt A, Corleis B, Kochmann J, Oliveira Esteves BI, Almeida L, Thomann L, Devisme C, Stalder H, Steiner S, Ochsenbein S, Schmied K, Labroussaa F, Jores J, V'kovski P, Cmiljanovic V, Alves MP, Benarafa C, Ebert N, Hoffmann D, Beer M, and Thiel V more...
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- Animals, Humans, Mice, Antibodies, Viral immunology, Antibodies, Viral blood, Female, Chlorocebus aethiops, Disease Models, Animal, Vero Cells, Antibodies, Neutralizing immunology, Vaccines, Attenuated immunology, Vaccines, Attenuated genetics, Vaccines, Attenuated administration & dosage, SARS-CoV-2 genetics, SARS-CoV-2 immunology, COVID-19 prevention & control, COVID-19 transmission, COVID-19 immunology, COVID-19 virology, COVID-19 Vaccines immunology, COVID-19 Vaccines administration & dosage, COVID-19 Vaccines genetics, Genome, Viral genetics
- Abstract
Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including 'one-to-stop' (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses., (© 2024. The Author(s).) more...
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- 2024
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8. Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation.
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Leborgne NG, Devisme C, Kozarac N, Berenguer Veiga I, Ebert N, Godel A, Grau-Roma L, Scherer M, Plattet P, Thiel V, Zimmer G, Taddeo A, and Benarafa C
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- Humans, Animals, Mice, Neutrophils metabolism, Spike Glycoprotein, Coronavirus, Inflammation, Serine Proteases metabolism, SARS-CoV-2 metabolism, COVID-19
- Abstract
Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases facilitate viral entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleaved the S protein at multiple sites and abrogated viral entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with viral clearance and inflammation in COVID-19 patients. more...
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- 2024
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9. 2023 International African Swine Fever Workshop: Critical Issues That Need to Be Addressed for ASF Control.
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Wang L, Ganges L, Dixon LK, Bu Z, Zhao D, Truong QL, Richt JA, Jin M, Netherton CL, Benarafa C, Summerfield A, Weng C, Peng G, Reis AL, Han J, Penrith ML, Mo Y, Su Z, Vu Hoang D, Pogranichniy RM, Balaban-Oglan DA, Li Y, Wang K, Cai X, and Shi J more...
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- Swine, Animals, Humans, Asia, China epidemiology, Africa epidemiology, Sus scrofa, Disease Outbreaks veterinary, African Swine Fever prevention & control, African Swine Fever epidemiology, African Swine Fever Virus
- Abstract
The 2023 International African Swine Fever Workshop (IASFW) took place in Beijing, China, on 18-20 September 2023. It was jointly organized by the U.S.-China Center for Animal Health (USCCAH) at Kansas State University (KSU) and the Chinese Veterinary Drug Association (CVDA) and sponsored by the United States Department of Agriculture Foreign Agricultural Service (USDA-FAS), Harbin Veterinary Research Institute, and Zoetis Inc. The objective of this workshop was to provide a platform for ASF researchers around the world to unite and share their knowledge and expertise on ASF control and prevention. A total of 24 outstanding ASF research scientists and experts from 10 countries attended this meeting. The workshop included presentations on current ASF research, opportunities for scientific collaboration, and discussions of lessons and experiences learned from China/Asia, Africa, and Europe. This article summarizes the meeting highlights and presents some critical issues that need to be addressed for ASF control and prevention in the future. more...
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- 2023
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10. Sex-specific differences in immune response to SARS-CoV-2 vaccination vanish with age.
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Brigger D, Guntern P, Jonsdottir HR, Pennington LF, Weber B, Taddeo A, Zimmer G, Leborgne NGF, Benarafa C, Jardetzky TS, and Eggel A
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- Female, Male, Humans, SARS-CoV-2, Vaccination, Immunity, COVID-19 Vaccines, COVID-19 prevention & control
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- 2023
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11. NET formation is independent of gasdermin D and pyroptotic cell death.
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Stojkov D, Claus MJ, Kozlowski E, Oberson K, Schären OP, Benarafa C, Yousefi S, and Simon HU
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- Mice, Animals, Intracellular Signaling Peptides and Proteins genetics, Intracellular Signaling Peptides and Proteins metabolism, Inflammasomes genetics, Inflammasomes metabolism, Cell Death, Neutrophils metabolism, Pyroptosis, Gasdermins
- Abstract
Neutrophil extracellular traps (NETs) are DNA scaffolds coated with granule proteins that are released by neutrophils to ensnare and kill bacteria. NET formation occurs in response to many stimuli through independent molecular pathways. Although NET release has been equated to a form of lytic cell death, live neutrophils can rapidly release antimicrobial NETs. Gasdermin D (GSDMD), which causes pyroptotic death in macrophages, is thought to be required for NET formation by neutrophils. Through experiments with known physiological activators of NET formation and ligands that activate canonical and noncanonical inflammasome signaling pathways, we demonstrated that Gsdmd -deficient mouse neutrophils were as competent as wild-type mouse neutrophils in producing NETs. Furthermore, GSDMD was not cleaved in wild-type neutrophils during NET release in response to inflammatory mediators. We found that activation of both canonical and noncanonical inflammasome signaling pathways resulted in GSDMD cleavage in wild-type neutrophils but was not associated with cell death. Moreover, NET formation as a result of either pathway of inflammasome activation did not require GSDMD. Together, these data suggest that NETs can be formed by viable neutrophils after inflammasome activation and that this function does not require GSDMD. more...
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- 2023
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12. The spike gene is a major determinant for the SARS-CoV-2 Omicron-BA.1 phenotype.
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Barut GT, Halwe NJ, Taddeo A, Kelly JN, Schön J, Ebert N, Ulrich L, Devisme C, Steiner S, Trüeb BS, Hoffmann B, Veiga IB, Leborgne NGF, Moreira EA, Breithaupt A, Wylezich C, Höper D, Wernike K, Godel A, Thomann L, Flück V, Stalder H, Brügger M, Esteves BIO, Zumkehr B, Beilleau G, Kratzel A, Schmied K, Ochsenbein S, Lang RM, Wider M, Machahua C, Dorn P, Marti TM, Funke-Chambour M, Rauch A, Widera M, Ciesek S, Dijkman R, Hoffmann D, Alves MP, Benarafa C, Beer M, and Thiel V more...
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- Animals, Cricetinae, Ferrets, Humans, Melphalan, Mice, Phenotype, RNA, Messenger, Spike Glycoprotein, Coronavirus genetics, gamma-Globulins, COVID-19, SARS-CoV-2 genetics
- Abstract
Variant of concern (VOC) Omicron-BA.1 has achieved global predominance in early 2022. Therefore, surveillance and comprehensive characterization of Omicron-BA.1 in advanced primary cell culture systems and animal models are urgently needed. Here, we characterize Omicron-BA.1 and recombinant Omicron-BA.1 spike gene mutants in comparison with VOC Delta in well-differentiated primary human nasal and bronchial epithelial cells in vitro, followed by in vivo fitness characterization in hamsters, ferrets and hACE2-expressing mice, and immunized hACE2-mice. We demonstrate a spike-mediated enhancement of early replication of Omicron-BA.1 in nasal epithelial cultures, but limited replication in bronchial epithelial cultures. In hamsters, Delta shows dominance over Omicron-BA.1, and in ferrets Omicron-BA.1 infection is abortive. In hACE2-knock-in mice, Delta and a Delta spike clone also show dominance over Omicron-BA.1 and an Omicron-BA.1 spike clone, respectively. Interestingly, in naïve K18-hACE2 mice, we observe Delta spike-mediated increased replication and pathogenicity and Omicron-BA.1 spike-mediated reduced replication and pathogenicity, suggesting that the spike gene is a major determinant of replication and pathogenicity. Finally, the Omicron-BA.1 spike clone is less well-controlled by mRNA-vaccination in K18-hACE2-mice and becomes more competitive compared to the progenitor and Delta spike clones, suggesting that spike gene-mediated immune evasion is another important factor that led to Omicron-BA.1 dominance., (© 2022. The Author(s).) more...
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- 2022
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13. The baseline immunological and hygienic status of pigs impact disease severity of African swine fever.
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Radulovic E, Mehinagic K, Wüthrich T, Hilty M, Posthaus H, Summerfield A, Ruggli N, and Benarafa C
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- Animals, Cytokines, Hygiene, Severity of Illness Index, Sus scrofa, Swine, African Swine Fever, African Swine Fever Virus genetics
- Abstract
African Swine Fever virus (ASFV) is a large double-enveloped DNA virus of the Asfarviridae family that causes a lethal hemorrhagic disease in domestic pigs and wild boars. Since 2007, a highly virulent genotype II strain has emerged and spread in Europe and South-East Asia, where millions of animals succumbed to the disease. Field- and laboratory-attenuated strains of ASFV cause highly variable clinical disease severity and survival, and mechanisms remain unclear. We hypothesized that the immunological and hygienic status of pigs is a determinant of ASF disease course. Here we compared the immunological profile at baseline and in response to ASFV infection in specific pathogen-free (SPF) and farm-raised Large White domestic pigs. At steady state, SPF pigs showed lower white blood cell counts and a lower basal inflammatory and antiviral transcriptomic profile compared to farm pigs, associated with profound differences in gut microbiome composition. After inoculation with a highly virulent ASFV genotype II strain (Armenia 2008), severe clinical signs, viremia and pro-inflammatory cytokines appeared sooner in SPF pigs, indicating a reduced capacity to control early virus replication. In contrast, during infection with an attenuated field isolate (Estonia 2014), SPF pigs presented a milder and shorter clinical disease with full recovery, whereas farm pigs presented severe protracted disease with 50% lethality. Interestingly, farm pigs showed higher production of inflammatory cytokines, whereas SPF pigs produced more anti-inflammatory IL-1ra early after infection and presented a stronger expansion of leukocytes in the recovery phase. Altogether, our data indicate that the hygiene-dependent innate immune status has a double-edge sword impact on immune responses in ASF pathogenesis. While the higher baseline innate immune activity helps the host in reducing initial virus replication, it promotes immunopathological cytokine responses, and delays lymphocyte proliferation after infection with an attenuated strain. Such effects should be considered for live vaccine development and vigilance., Competing Interests: The authors have declared that no competing interests exist. more...
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- 2022
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14. Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2.
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Taddeo A, Veiga IB, Devisme C, Boss R, Plattet P, Weigang S, Kochs G, Thiel V, Benarafa C, and Zimmer G
- Abstract
Immunization with vesicular stomatitis virus (VSV)-vectored COVID-19 vaccine candidates expressing the SARS-CoV-2 spike protein in place of the VSV glycoprotein relies implicitly on expression of the ACE2 receptor at the muscular injection site. Here, we report that such a viral vector vaccine did not induce protective immunity following intramuscular immunization of K18-hACE2 transgenic mice. However, when the viral vector was trans-complemented with the VSV glycoprotein, intramuscular immunization resulted in high titers of spike-specific neutralizing antibodies. The vaccinated animals were fully protected following infection with a lethal dose of SARS-CoV-2-S
D614G via the nasal route, and partially protected if challenged with the SARS-CoV-2Delta variant. While dissemination of the challenge virus to the brain was completely inhibited, replication in the lung with consequent lung pathology was not entirely controlled. Thus, intramuscular immunization was clearly enhanced by trans-complementation of the VSV-vectored vaccines by the VSV glycoprotein and led to protection from COVID-19, although not achieving sterilizing immunity., (© 2022. The Author(s).) more...- Published
- 2022
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15. Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.
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Ulrich L, Halwe NJ, Taddeo A, Ebert N, Schön J, Devisme C, Trüeb BS, Hoffmann B, Wider M, Fan X, Bekliz M, Essaidi-Laziosi M, Schmidt ML, Niemeyer D, Corman VM, Kraft A, Godel A, Laloli L, Kelly JN, Calderon BM, Breithaupt A, Wylezich C, Berenguer Veiga I, Gultom M, Osman S, Zhou B, Adea K, Meyer B, Eberhardt CS, Thomann L, Gsell M, Labroussaa F, Jores J, Summerfield A, Drosten C, Eckerle IA, Wentworth DE, Dijkman R, Hoffmann D, Thiel V, Beer M, and Benarafa C more...
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- Amino Acid Substitution, Angiotensin-Converting Enzyme 2 genetics, Angiotensin-Converting Enzyme 2 metabolism, Animals, Animals, Laboratory virology, COVID-19 veterinary, Cricetinae, Disease Models, Animal, Epithelial Cells virology, Female, Ferrets virology, Humans, Male, Mesocricetus virology, Mice, Mice, Transgenic, SARS-CoV-2 genetics, SARS-CoV-2 growth & development, Spike Glycoprotein, Coronavirus genetics, Spike Glycoprotein, Coronavirus metabolism, Virulence genetics, COVID-19 transmission, COVID-19 virology, Mutation, SARS-CoV-2 classification, SARS-CoV-2 physiology, Virus Replication
- Abstract
Emerging variants of concern (VOCs) are driving the COVID-19 pandemic
1,2 . Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3 . Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G ) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals., (© 2021. The Author(s).) more...- Published
- 2022
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16. Granule Leakage Induces Cell-Intrinsic, Granzyme B-Mediated Apoptosis in Mast Cells.
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Burgener SS, Brügger M, Leborgne NGF, Sollberger S, Basilico P, Kaufmann T, Bird PI, and Benarafa C
- Abstract
Mast cells are multifunctional immune cells scattered in tissues near blood vessels and mucosal surfaces where they mediate important reactions against parasites and contribute to the pathogenesis of allergic reactions. Serine proteases released from secretory granules upon mast cell activation contribute to these functions by modulating cytokine activity, platelet activation and proteolytic neutralization of toxins. The forced release of granule proteases into the cytosol of mast cells to induce cell suicide has recently been proposed as a therapeutic approach to reduce mast cell numbers in allergic diseases, but the molecular pathways involved in granule-mediated mast cell suicide are incompletely defined. To identify intrinsic granule proteases that can cause mast cell death, we used mice deficient in cytosolic serine protease inhibitors and their respective target proteases. We found that deficiency in Serpinb1a, Serpinb6a, and Serpinb9a or in their target proteases did not alter the kinetics of apoptosis induced by growth factor deprivation in vitro or the number of peritoneal mast cells in vivo . The serine protease cathepsin G induced marginal cell death upon mast cell granule permeabilization only when its inhibitors Serpinb1a or Serpinb6a were deleted. In contrast, the serine protease granzyme B was essential for driving apoptosis in mast cells. On granule permeabilization, granzyme B was required for caspase-3 processing and cell death. Moreover, cytosolic granzyme B inhibitor Serpinb9a prevented caspase-3 processing and mast cell death in a granzyme B-dependent manner. Together, our findings demonstrate that cytosolic serpins provide an inhibitory shield preventing granule protease-induced mast cell apoptosis, and that the granzyme B-Serpinb9a-caspase-3 axis is critical in mast cell survival and could be targeted in the context of allergic diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Burgener, Brügger, Leborgne, Sollberger, Basilico, Kaufmann, Bird and Benarafa.) more...
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- 2021
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17. ATG5 promotes eosinopoiesis but inhibits eosinophil effector functions.
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Germic N, Hosseini A, Stojkov D, Oberson K, Claus M, Benarafa C, Calzavarini S, Angelillo-Scherrer A, Arnold IC, Müller A, Riether C, Yousefi S, and Simon HU
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- Animals, Autophagy-Related Protein 5 biosynthesis, Autophagy-Related Protein 5 deficiency, Autophagy-Related Protein 5 genetics, Bone Marrow pathology, CRISPR-Cas Systems, Cell Degranulation, Cell Line, Tumor, Cells, Cultured, Citrobacter rodentium, Colony-Forming Units Assay, Enterobacteriaceae Infections immunology, Eosinophils cytology, Eosinophils immunology, Humans, Hypereosinophilic Syndrome blood, Hypereosinophilic Syndrome pathology, Interleukin-5 genetics, Leukocyte Count, MAP Kinase Signaling System genetics, Mice, Mice, Knockout, Mice, Transgenic, Oncogene Proteins, Fusion genetics, Receptor, Platelet-Derived Growth Factor alpha genetics, mRNA Cleavage and Polyadenylation Factors genetics, Autophagy-Related Protein 5 physiology, Eosinophils physiology, Myelopoiesis physiology
- Abstract
Eosinophils are white blood cells that contribute to the regulation of immunity and are involved in the pathogenesis of numerous inflammatory diseases. In contrast to other cells of the immune system, no information is available regarding the role of autophagy in eosinophil differentiation and functions. To study the autophagic pathway in eosinophils, we generated conditional knockout mice in which Atg5 is deleted within the eosinophil lineage only (designated Atg5eoΔ mice). Eosinophilia was provoked by crossbreeding Atg5eoΔ mice with Il5 (IL-5) overexpressing transgenic mice (designated Atg5eoΔIl5tg mice). Deletion of Atg5 in eosinophils resulted in a dramatic reduction in the number of mature eosinophils in blood and an increase of immature eosinophils in the bone marrow. Atg5-knockout eosinophil precursors exhibited reduced proliferation under both in vitro and in vivo conditions but no increased cell death. Moreover, reduced differentiation of eosinophils in the absence of Atg5 was also observed in mouse and human models of chronic eosinophilic leukemia. Atg5-knockout blood eosinophils exhibited augmented levels of degranulation and bacterial killing in vitro. Moreover, in an experimental in vivo model, we observed that Atg5eoΔ mice achieve better clearance of the local and systemic bacterial infection with Citrobacter rodentium. Evidence for increased degranulation of ATG5low-expressing human eosinophils was also obtained in both tissues and blood. Taken together, mouse and human eosinophil hematopoiesis and effector functions are regulated by ATG5, which controls the amplitude of overall antibacterial eosinophil immune responses., (© 2021 by The American Society of Hematology.) more...
- Published
- 2021
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18. The specific features of the developing T cell compartment of the neonatal lung are a determinant of respiratory syncytial virus immunopathogenesis.
- Author
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Démoulins T, Brügger M, Zumkehr B, Oliveira Esteves BI, Mehinagic K, Fahmi A, Borcard L, Taddeo A, Jandrasits D, Posthaus H, Benarafa C, Ruggli N, and Alves MP
- Subjects
- Animals, Animals, Newborn, Cell Differentiation immunology, Cells, Cultured, Child, Preschool, Disease Models, Animal, Disease Progression, Humans, Lung growth & development, Lung pathology, Lung virology, Respiratory Syncytial Virus Infections congenital, Respiratory Syncytial Virus Infections pathology, Respiratory Tract Infections congenital, Respiratory Tract Infections pathology, Sheep growth & development, Sheep immunology, T-Lymphocytes immunology, T-Lymphocytes physiology, Lung immunology, Respiratory Syncytial Virus Infections immunology, Respiratory Tract Infections immunology, T-Lymphocytes pathology
- Abstract
The human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants, possibly due to the properties of the immature neonatal pulmonary immune system. Using the newborn lamb, a classical model of human lung development and a translational model of RSV infection, we aimed to explore the role of cell-mediated immunity in RSV disease during early life. Remarkably, in healthy conditions, the developing T cell compartment of the neonatal lung showed major differences to that seen in the mature adult lung. The most striking observation being a high baseline frequency of bronchoalveolar IL-4-producing CD4+ and CD8+ T cells, which declined progressively over developmental age. RSV infection exacerbated this pro-type 2 environment in the bronchoalveolar space, rather than inducing a type 2 response per se. Moreover, regulatory T cell suppressive functions occurred very early to dampen this pro-type 2 environment, rather than shutting them down afterwards, while γδ T cells dropped and failed to produce IL-17. Importantly, RSV disease severity was related to the magnitude of those unconventional bronchoalveolar T cell responses. These findings provide novel insights in the mechanisms of RSV immunopathogenesis in early life, and constitute a major step for the understanding of RSV disease severity., Competing Interests: The authors have declared that no competing interests exist. more...
- Published
- 2021
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19. SARS-CoV-2 spike D614G change enhances replication and transmission.
- Author
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Zhou B, Thao TTN, Hoffmann D, Taddeo A, Ebert N, Labroussaa F, Pohlmann A, King J, Steiner S, Kelly JN, Portmann J, Halwe NJ, Ulrich L, Trüeb BS, Fan X, Hoffmann B, Wang L, Thomann L, Lin X, Stalder H, Pozzi B, de Brot S, Jiang N, Cui D, Hossain J, Wilson MM, Keller MW, Stark TJ, Barnes JR, Dijkman R, Jores J, Benarafa C, Wentworth DE, Thiel V, and Beer M more...
- Subjects
- Angiotensin-Converting Enzyme 2 genetics, Angiotensin-Converting Enzyme 2 metabolism, Animals, Bronchi cytology, Bronchi virology, COVID-19 epidemiology, Cell Line, Cells, Cultured, Cricetinae, Disease Models, Animal, Epithelial Cells virology, Female, Ferrets virology, Founder Effect, Gene Knock-In Techniques, Genetic Fitness, Humans, Male, Mesocricetus, Mice, Nasal Mucosa cytology, Nasal Mucosa virology, Protein Binding, RNA, Viral analysis, Receptors, Coronavirus metabolism, SARS-CoV-2 metabolism, SARS-CoV-2 pathogenicity, COVID-19 transmission, COVID-19 virology, Mutation, SARS-CoV-2 genetics, SARS-CoV-2 physiology, Spike Glycoprotein, Coronavirus genetics, Virus Replication genetics
- Abstract
During the evolution of SARS-CoV-2 in humans, a D614G substitution in the spike glycoprotein (S) has emerged; virus containing this substitution has become the predominant circulating variant in the COVID-19 pandemic
1 . However, whether the increasing prevalence of this variant reflects a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains unknown. Here we use isogenic SARS-CoV-2 variants to demonstrate that the variant that contains S(D614G) has enhanced binding to the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2), increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a human ACE2 knock-in mouse model, and markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Our data show that the D614G substitution in S results in subtle increases in binding and replication in vitro, and provides a real competitive advantage in vivo-particularly during the transmission bottleneck. Our data therefore provide an explanation for the global predominance of the variant that contains S(D614G) among the SARS-CoV-2 viruses that are currently circulating. more...- Published
- 2021
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20. Chronic cigarette smoke exposure and pneumococcal infection induce oropharyngeal microbiota dysbiosis and contribute to long-lasting lung damage in mice.
- Author
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Hilty M, Wüthrich TM, Godel A, Adelfio R, Aebi S, Burgener SS, Illgen-Wilcke B, and Benarafa C
- Subjects
- Animals, Bacteria drug effects, Bacteria genetics, Bacteria isolation & purification, DNA, Ribosomal genetics, Disease Models, Animal, Disease Progression, Dysbiosis chemically induced, Dysbiosis complications, Dysbiosis microbiology, Female, Longitudinal Studies, Mice, Phylogeny, Pulmonary Emphysema chemically induced, Pulmonary Emphysema microbiology, RNA, Bacterial genetics, Sequence Analysis, DNA methods, Tobacco Products adverse effects, Bacteria classification, Dysbiosis etiology, Oropharynx microbiology, Pneumococcal Infections microbiology, Pulmonary Emphysema genetics, RNA, Ribosomal, 16S genetics, Smoke adverse effects
- Abstract
Environmental factors, such as cigarette smoking or lung infections, may influence chronic obstructive pulmonary disease (COPD) progression by modifying the respiratory tract microbiome. However, whether the disease itself induces or maintains dysbiosis remains undefined. In this longitudinal study, we investigated the oropharyngeal microbiota composition and disease progression of mice (in cages of 5-10 mice per cage) before, during and up to 3 months after chronic cigarette smoke exposure or exposure to room air for 6 months. Cigarette smoke exposure induced pulmonary emphysema measurable at the end of exposure for 6 months, as well as 3 months following smoke exposure cessation. Using both classical culture methods and 16S rRNA sequencing, we observed that cigarette smoke exposure altered the relative composition of the oropharyngeal microbiota and reduced its diversity ( P <0.001). More than 60 taxa were substantially reduced after 6 months of smoke exposure ( P <0.001) However, oropharyngeal microbiota disordering was reversed 3 months after smoke exposure cessation and no significant difference was observed compared to age-matched control mice. The effects of lung infection with Streptococcus pneumoniae on established smoke-induced emphysema and on the oropharyngeal microbiota were also evaluated. Inoculation with S. pneumoniae induced lung damage and altered the microbiota composition for a longer time compared to control groups infected but not previously exposed to smoke ( P =0.01). Our data demonstrate effects of cigarette smoke and pneumococcus infection leading to altered microbiota and emphysema development. The reversal of the disordering of the microbiota composition, but not lung damage, following smoke exposure cessation and after clearance of infection suggest that changes in lung structure are not sufficient to sustain a disordered microbiota in mice. Whether changes in the airway microbiota contribute to inducing emphysema requires further investigation. more...
- Published
- 2020
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21. Serpinb1a Is Dispensable for the Development and Cytokine Response of Invariant Natural Killer T Cell Subsets.
- Author
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Leborgne NGF, Taddeo A, Freigang S, and Benarafa C
- Subjects
- Animals, Dendritic Cells immunology, Female, Galactosylceramides adverse effects, Homeostasis genetics, Inflammation chemically induced, Inflammation immunology, Inflammation metabolism, Liver immunology, Lung immunology, Lymphocyte Activation drug effects, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Animal, Neutrophils immunology, Serpins genetics, Signal Transduction drug effects, Spleen immunology, Cell Differentiation genetics, Cytokines metabolism, Lymphocyte Activation genetics, Natural Killer T-Cells immunology, Serpins deficiency, Signal Transduction genetics
- Abstract
Invariant natural killer T (iNKT) cells are innate-like T lymphocytes. They quickly respond to antigenic stimulation by producing copious amounts of cytokines and chemokines. iNKT precursors differentiate into three subsets iNKT1, iNKT2, and iNKT17 with specific cytokine production signatures. While key transcription factors drive subset differentiation, factors that regulate iNKT subset homeostasis remain incompletely defined. Transcriptomic analyses of thymic iNKT subsets indicate that Serpinb1a is one of the most specific transcripts for iNKT17 cells suggesting that iNKT cell maintenance and function may be regulated by Serpinb1a. Serpinb1a is a major survival factor in neutrophils and prevents cell death in a cell-autonomous manner. It also controls inflammation in models of bacterial and viral infection as well as in LPS-driven inflammation. Here, we examined the iNKT subsets in neutropenic Serpinb1a
-/- mice as well as in Serpinb1a-/- mice with normal neutrophil counts due to transgenic re-expression of SERPINB1 in neutrophils. In steady state, we found no significant effect of Serpinb1a -deficiency on the proliferation and numbers of iNKT subsets in thymus, lymph nodes, lung, liver and spleen. Following systemic activation with α-galactosylceramide, the prototypic glycolipid agonist of iNKT cells, we observed similar serum levels of IFN-γ and IL-4 between genotypes. Moreover, splenic dendritic cells showed normal upregulation of maturation markers following iNKT cell activation with α-galactosylceramide. Finally, lung instillation of α-galactosylceramide induced a similar recruitment of neutrophils and production of iNKT-derived cytokines IL-17, IFN-γ, and IL-4 in wild-type and Serpinb1a-/- mice. Taken together, our results indicate that Serpinb1a, while dominantly expressed in iNKT17 cells, is not essential for iNKT cell homeostasis, subset differentiation and cytokine release., (Copyright © 2020 Leborgne, Taddeo, Freigang and Benarafa.) more...- Published
- 2020
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22. SARS-CoV-2 spike D614G variant confers enhanced replication and transmissibility.
- Author
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Zhou B, Thao TTN, Hoffmann D, Taddeo A, Ebert N, Labroussaa F, Pohlmann A, King J, Portmann J, Halwe NJ, Ulrich L, Trüeb BS, Kelly JN, Fan X, Hoffmann B, Steiner S, Wang L, Thomann L, Lin X, Stalder H, Pozzi B, de Brot S, Jiang N, Cui D, Hossain J, Wilson M, Keller M, Stark TJ, Barnes JR, Dijkman R, Jores J, Benarafa C, Wentworth DE, Thiel V, and Beer M more...
- Abstract
During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic
1perscript> . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating. more... - Published
- 2020
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23. Correction: Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.
- Author
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Ohayon D, De Chiara A, My-Chan Dang P, Thieblemont N, Chatfield S, Marzaioli V, Burgener SS, Mocek J, Candalh C, Pintard C, Tacnet-Delorme P, Renault G, Lagoutte I, Favier M, Walker F, Hurtado-Nedelec M, Desplancq D, Weiss E, Benarafa C, Housset D, Marie JC, Frachet P, El-Benna J, and Witko-Sarsat V more...
- Published
- 2019
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24. Cytosolic PCNA interacts with p47phox and controls NADPH oxidase NOX2 activation in neutrophils.
- Author
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Ohayon D, De Chiara A, Dang PM, Thieblemont N, Chatfield S, Marzaioli V, Burgener SS, Mocek J, Candalh C, Pintard C, Tacnet-Delorme P, Renault G, Lagoutte I, Favier M, Walker F, Hurtado-Nedelec M, Desplancq D, Weiss E, Benarafa C, Housset D, Marie JC, Frachet P, El-Benna J, and Witko-Sarsat V more...
- Subjects
- Animals, Cell Line, Tumor, Cells, Cultured, Colitis chemically induced, Colitis prevention & control, Enzyme Activation drug effects, Female, Humans, Male, Mice, Inbred C57BL, Mice, Knockout, NADPH Oxidases genetics, Protein Binding, Reactive Oxygen Species metabolism, Small Molecule Libraries pharmacology, Trinitrobenzenesulfonic Acid, Cytosol metabolism, NADPH Oxidase 2 metabolism, NADPH Oxidases metabolism, Neutrophils metabolism, Proliferating Cell Nuclear Antigen metabolism
- Abstract
Neutrophils produce high levels of reactive oxygen species (ROS) by NADPH oxidase that are crucial for host defense but can lead to tissue injury when produced in excess. We previously described that proliferating cell nuclear antigen (PCNA), a nuclear scaffolding protein pivotal in DNA synthesis, controls neutrophil survival through its cytosolic association with procaspases. We herein showed that PCNA associated with p47phox, a key subunit of NADPH oxidase, and that this association regulated ROS production. Surface plasmon resonance and crystallography techniques demonstrated that the interdomain-connecting loop of PCNA interacted directly with the phox homology ( PX) domain of the p47phox. PCNA inhibition by competing peptides or by T2AA, a small-molecule PCNA inhibitor, decreased NADPH oxidase activation in vitro. Furthermore, T2AA provided a therapeutic benefit in mice during trinitro-benzene-sulfonic acid (TNBS)-induced colitis by decreasing oxidative stress, accelerating mucosal repair, and promoting the resolution of inflammation. Our data suggest that targeting PCNA in inflammatory neutrophils holds promise as a multifaceted antiinflammatory strategy., (© 2019 Ohayon et al.) more...
- Published
- 2019
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25. Cathepsin G Inhibition by Serpinb1 and Serpinb6 Prevents Programmed Necrosis in Neutrophils and Monocytes and Reduces GSDMD-Driven Inflammation.
- Author
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Burgener SS, Leborgne NGF, Snipas SJ, Salvesen GS, Bird PI, and Benarafa C
- Subjects
- Animals, Apoptosis, Endotoxins toxicity, Female, Inflammasomes, Inflammation chemically induced, Inflammation metabolism, Inflammation pathology, Intracellular Signaling Peptides and Proteins genetics, Macrophages, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Monocytes metabolism, Necrosis, Neutrophils metabolism, Phosphate-Binding Proteins genetics, Pyroptosis, Cathepsin G antagonists & inhibitors, Inflammation prevention & control, Intracellular Signaling Peptides and Proteins metabolism, Monocytes pathology, Neutrophils pathology, Phosphate-Binding Proteins metabolism, Serpins physiology
- Abstract
Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a
-/- mice. Furthermore, Sb1a.Sb6a-/- mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a-/- macrophages showed increased IL-1β release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.) more...- Published
- 2019
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26. The Genetic Background of Mice Influences the Effects of Cigarette Smoke on Onset and Severity of Experimental Autoimmune Encephalomyelitis.
- Author
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Enzmann G, Adelfio R, Godel A, Haghayegh Jahromi N, Tietz S, Burgener SS, Deutsch U, Wekerle H, Benarafa C, and Engelhardt B
- Subjects
- Age of Onset, Animals, Biopsy, Brain metabolism, Brain pathology, Disease Models, Animal, Disease Susceptibility, Encephalomyelitis, Autoimmune, Experimental metabolism, Immunohistochemistry, Mice, Multiple Sclerosis diagnosis, Multiple Sclerosis etiology, Multiple Sclerosis metabolism, Phenotype, Risk Assessment, Risk Factors, Severity of Illness Index, Spinal Cord metabolism, Spinal Cord pathology, Encephalomyelitis, Autoimmune, Experimental diagnosis, Encephalomyelitis, Autoimmune, Experimental etiology, Genetic Background, Smoking adverse effects
- Abstract
Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followed by a protracted disease course in SJL/J mice. In contrast, cigarette smoke exposure ameliorated the EAE clinical score in C57BL/6J mice. Our exploratory studies therefore show that genetic background influences the effects of cigarette smoke on autoimmune neuroinflammation. Importantly, our findings expose the challenge of identifying an animal model for studying the influence of cigarette smoke in MS. more...
- Published
- 2019
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27. Untangling "NETosis" from NETs.
- Author
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Yousefi S, Stojkov D, Germic N, Simon D, Wang X, Benarafa C, and Simon HU
- Subjects
- Cell Death, Humans, Necrosis, Extracellular Traps metabolism, Neutrophil Activation, Neutrophils metabolism
- Abstract
Neutrophil extracellular trap (NET) formation is a cellular function of neutrophils that facilitates the immobilization and killing of invading microorganisms in the extracellular milieu. To form NETs, neutrophils release a DNA scaffold consisting of mitochondrial DNA binding granule proteins. This process does not depend on cell death, but requires glycolytic ATP production for rearrangements in the microtubule network and F-actin. Such cytoskeletal rearrangements are essential for both mitochondrial DNA release and degranulation. However, the formation of NETs has also been described as a distinct form of programed, necrotic cell death, a process designated "NETosis." Necrotic cell death of neutrophils is associated with the permeabilization of both plasma and nuclear membranes resulting in a kind of extracellular cloud of nuclear DNA. The molecular mechanisms eliciting necrotic neutrophil death have been investigated and appear to be different from those responsible for NET formation following mitochondrial DNA release. Here, we discriminate between the mechanisms responsible for the release of mitochondrial versus nuclear DNA and address their respective functions. Our aim is to clarify existing differences of opinion in the fields of NET formation and neutrophil death., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.) more...
- Published
- 2019
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28. Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production.
- Author
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Amini P, Stojkov D, Felser A, Jackson CB, Courage C, Schaller A, Gelman L, Soriano ME, Nuoffer JM, Scorrano L, Benarafa C, Yousefi S, and Simon HU
- Subjects
- Actins metabolism, Animals, Anti-Bacterial Agents pharmacology, Bone Marrow, Cell Line, Tumor, Electron Transport Complex I drug effects, Electron Transport Complex I metabolism, GTP Phosphohydrolases genetics, Gene Expression Profiling, Humans, Immunity, Innate, Lung immunology, Lung microbiology, Mice, Mice, Knockout, Microtubules metabolism, Mitochondria genetics, Mitochondria metabolism, Mitochondrial Membranes metabolism, Neutrophils cytology, Pseudomonas Infections immunology, Pseudomonas aeruginosa pathogenicity, Reactive Oxygen Species metabolism, Adenosine Triphosphate metabolism, Extracellular Traps metabolism, GTP Phosphohydrolases immunology, GTP Phosphohydrolases metabolism, Glycolysis physiology, Neutrophils metabolism
- Abstract
Optic atrophy 1 (OPA1) is a mitochondrial inner membrane protein that has an important role in mitochondrial fusion and structural integrity. Dysfunctional OPA1 mutations cause atrophy of the optic nerve leading to blindness. Here, we show that OPA1 has an important role in the innate immune system. Using conditional knockout mice lacking Opa1 in neutrophils (Opa1
N∆ ), we report that lack of OPA1 reduces the activity of mitochondrial electron transport complex I in neutrophils. This then causes a decline in adenosine-triphosphate (ATP) production through glycolysis due to lowered NAD+ availability. Additionally, we show that OPA1-dependent ATP production in these cells is required for microtubule network assembly and for the formation of neutrophil extracellular traps. Finally, we show that Opa1N∆ mice exhibit a reduced antibacterial defense capability against Pseudomonas aeruginosa. more...- Published
- 2018
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29. Transgenic Mice Expressing Human Proteinase 3 Exhibit Sustained Neutrophil-Associated Peritonitis.
- Author
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Martin KR, Pederzoli-Ribeil M, Pacreau E, Burgener SS, Dahdah A, Candalh C, Lauret E, Foretz M, Mouthon L, Lucas B, Thieblemont N, Benarafa C, Launay P, and Witko-Sarsat V
- Subjects
- Animals, Annexin A1 metabolism, Apoptosis, Disease Models, Animal, Humans, Mice, Mice, Inbred C57BL, Mice, Transgenic, Myeloblastin genetics, Peritonitis chemically induced, Phagocytosis, Sepsis chemically induced, Zymosan, Myeloblastin metabolism, Neutrophils immunology, Peritoneal Cavity pathology, Peritonitis immunology, Sepsis immunology
- Abstract
Proteinase 3 (PR3) is a myeloid serine protease expressed in neutrophils, monocytes, and macrophages. PR3 has a number of well-characterized proinflammatory functions, including cleaving and activating chemokines and controlling cell survival and proliferation. When presented on the surface of apoptotic neutrophils, PR3 can disrupt the normal anti-inflammatory reprogramming of macrophages following the phagocytosis of apoptotic cells. To better understand the function of PR3 in vivo, we generated a human PR3 transgenic mouse (hPR3Tg). During zymosan-induced peritonitis, hPR3Tg displayed an increased accumulation of neutrophils within the peritoneal cavity compared with wild-type control mice, with no difference in the recruitment of macrophages or B or T lymphocytes. Mice were also subjected to cecum ligation and puncture, a model used to induce peritoneal inflammation through infection. hPR3Tg displayed decreased survival rates in acute sepsis, associated with increased neutrophil extravasation. The decreased survival and increased neutrophil accumulation were associated with the cleavage of annexin A1, a powerful anti-inflammatory protein known to facilitate the resolution of inflammation. Additionally, neutrophils from hPR3Tg displayed enhanced survival during apoptosis compared with controls, and this may also contribute to the increased accumulation observed during the later stages of inflammation. Taken together, our data suggest that human PR3 plays a proinflammatory role during acute inflammatory responses by affecting neutrophil accumulation, survival, and the resolution of inflammation., (Copyright © 2017 by The American Association of Immunologists, Inc.) more...
- Published
- 2017
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30. Role of granule proteases in the life and death of neutrophils.
- Author
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Benarafa C and Simon HU
- Subjects
- Animals, Apoptosis physiology, Cell Death physiology, Cytoplasmic Granules enzymology, Feedback, Physiological, Humans, Leukocyte Elastase genetics, Mutation, Myelopoiesis physiology, Neutropenia etiology, Neutrophils immunology, Serpins metabolism, Neutrophils cytology, Neutrophils enzymology, Peptide Hydrolases metabolism
- Abstract
Neutrophils constitute a crucial component of the innate immune defenses against microbes. Produced in the bone marrow and patrolling in blood vessels, neutrophils are recruited to injured tissues and are immediately active to contain pathogen invasion. Neutrophils undergo programmed cell death by multiple, context-specific pathways, which have consequences on immunopathology and disease outcome. Studies in the last decade indicate additional functions for neutrophils - or a subset of neutrophils - in modulating adaptive responses and tumor progression. Neutrophil granules contain abundant amounts of various proteases, which are directly implicated in protective and pathogenic functions of neutrophils. It now emerges that neutral serine proteases such as cathepsin G and proteinase-3 also contribute to the neutrophil life cycle, but do so via different pathways than that of the aspartate protease cathepsin D and that of mutants of the serine protease elastase. The aim of this review is to appraise the present knowledge of the function of neutrophil granule proteases and their inhibitors in neutrophil cell death, and to integrate these findings in the current understandings of neutrophil life cycle and programmed cell death pathways., (Copyright © 2016 Elsevier Inc. All rights reserved.) more...
- Published
- 2017
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31. RhoH is a negative regulator of eosinophilopoiesis.
- Author
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Stoeckle C, Geering B, Yousefi S, Rožman S, Andina N, Benarafa C, and Simon HU
- Subjects
- Animals, Bone Marrow metabolism, Cell Count, Cell Cycle, Cell Differentiation, Cell Movement, Cell Survival, Cells, Cultured, Chimera, Eosinophils cytology, GATA2 Transcription Factor metabolism, Humans, Immunophenotyping, Interleukin-5 metabolism, Longevity, Mice, Inbred C57BL, Mice, Knockout, Phenotype, Receptors, Interleukin-5 metabolism, Up-Regulation, Eosinophils metabolism, Transcription Factors metabolism, rho GTP-Binding Proteins metabolism
- Abstract
Eosinophils are frequently elevated in pathological conditions and can cause tissue damage and disease exacerbation. The number of eosinophils in the blood is largely regulated by factors controlling their production in the bone marrow. While several exogenous factors, such as interleukin-5, have been described to promote eosinophil differentiation, comparatively little is known about eosinophil-intrinsic factors that control their de novo generation. Here, we report that the small atypical GTPase RhoH is induced during human eosinophil differentiation, highly expressed in mature blood eosinophils and further upregulated in patients suffering from a hypereosinophilic syndrome. Overexpression of RhoH increases, in a Rho-associated protein kinase-dependent manner, the expression of GATA-2, a transcription factor involved in regulating eosinophil differentiation. In RhoH
-/- mice, we observed reduced GATA-2 expression as well as accelerated eosinophil differentiation both in vitro and in vivo. Conversely, RhoH overexpression in bone marrow progenitors reduces eosinophil development in mixed bone marrow chimeras. These results highlight a novel negative regulatory role for RhoH in eosinophil differentiation, most likely in consequence of altered GATA-2 levels. more...- Published
- 2016
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32. Adamts18 deletion results in distinct developmental defects and provides a model for congenital disorders of lens, lung, and female reproductive tract development.
- Author
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Ataca D, Caikovski M, Piersigilli A, Moulin A, Benarafa C, Earp SE, Guri Y, Kostic C, Arsenijevic Y, Soininen R, Apte SS, and Brisken C
- Abstract
The ADAMTS family comprises 19 secreted metalloproteinases that cleave extracellular matrix components and have diverse functions in numerous disease and physiological contexts. A number of them remain 'orphan' proteases and among them is ADAMTS18, which has been implicated in developmental eye disorders, platelet function and various malignancies. To assess in vivo function of ADAMTS18, we generated a mouse strain with inactivated Adamts18 alleles. In the C57Bl6/Ola background, Adamts18-deficient mice are born in a normal Mendelian ratio, and are viable but show a transient growth delay. Histological examination revealed a 100% penetrant eye defect resulting from leakage of lens material through the lens capsule occurring at embryonic day (E)13.5, when the lens grows rapidly. Adamts18-deficient lungs showed altered bronchiolar branching. Fifty percent of mutant females are infertile because of vaginal obstruction due to either a dorsoventral vaginal septum or imperforate vagina. The incidence of ovarian rete is increased in the mutant mouse strain. Thus, Adamts18 is essential in the development of distinct tissues and the new mouse strain is likely to be useful for investigating ADAMTS18 function in human disease, particularly in the contexts of infertility and carcinogenesis., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.) more...
- Published
- 2016
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33. Exogenous cathepsin G upregulates cell surface MHC class I molecules on immune and glioblastoma cells.
- Author
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Giese M, Turiello N, Molenda N, Palesch D, Meid A, Schroeder R, Basilico P, Benarafa C, Halatsch ME, Zimecki M, Westhoff MA, Wirtz CR, and Burster T
- Subjects
- Animals, Cathepsin G genetics, Cathepsin G metabolism, Cell Line, Cell Line, Tumor, Cell Membrane metabolism, Disease Models, Animal, Female, Gene Expression Regulation, Neoplastic drug effects, Glioblastoma immunology, Histocompatibility Antigens Class I genetics, Humans, Immune System immunology, Lactoferrin metabolism, Male, Mice, Mice, Knockout, Monocytes drug effects, Monocytes metabolism, Neoplastic Stem Cells drug effects, Neoplastic Stem Cells metabolism, Proteolysis, Cathepsin G pharmacology, Glioblastoma genetics, Glioblastoma metabolism, Histocompatibility Antigens Class I metabolism, Immune System cytology, Immune System metabolism
- Abstract
Major histocompatibility complex (MHC) class I molecules present antigenic peptides to cytotoxic T cells. During an adaptive immune response, MHC molecules are regulated by several mechanisms including lipopolysaccharide (LPS) and interferon gamma (IFN-g). However, it is unclear whether the serine protease cathepsin G (CatG), which is generally secreted by neutrophils at the site of inflammation, might regulate MHC I molecules. We identified CatG, and to a higher extend CatG and lactoferrin (LF), as an exogenous regulator of cell surface MHC I expression of immune cells and glioblastoma stem cells. In addition, levels of MHC I molecules are reduced on dendritic cells from CatG deficient mice compared to their wild type counterparts. Furthermore, cell surface CatG on immune cells, including T cells, B cells, and NK cells triggers MHC I on THP-1 monocytes suggesting a novel mechanism for CatG to facilitate intercellular communication between infiltrating cells and the respective target cell. Subsequently, our findings highlight the pivotal role of CatG as a checkpoint protease which might force target cells to display their intracellular MHC I:antigen repertoire. more...
- Published
- 2016
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34. Distinct gene expression patterns correlate with developmental and functional traits of iNKT subsets.
- Author
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Georgiev H, Ravens I, Benarafa C, Förster R, and Bernhardt G
- Subjects
- Animals, Biomarkers metabolism, Cell Movement, Cytokines metabolism, Female, Lung cytology, Mice, Inbred BALB C, Mice, Inbred C57BL, Natural Killer T-Cells cytology, Phenotype, Receptors, Antigen, T-Cell metabolism, Receptors, Chemokine metabolism, Serpins deficiency, Serpins metabolism, Signal Transduction, Thymus Gland cytology, Transcriptome genetics, Gene Expression Profiling, Gene Expression Regulation, Natural Killer T-Cells metabolism, T-Lymphocyte Subsets metabolism
- Abstract
Invariant natural killer T (iNKT) cells comprise a subpopulation of innate lymphocytes developing in thymus. A new model proposes subdividing murine iNKT cells into iNKT1, 2 and 17 cells. Here, we use transcriptome analyses of iNKT1, 2 and 17 subsets isolated from BALB/c and C57BL/6 thymi to identify candidate genes that may affect iNKT cell development, migration or function. We show that Fcɛr1γ is involved in generation of iNKT1 cells and that SerpinB1 modulates frequency of iNKT17 cells. Moreover, a considerable proportion of iNKT17 cells express IL-4 and IL-17 simultaneously. The results presented not only validate the usefulness of the iNKT1/2/17-concept but also provide new insights into iNKT cell biology. more...
- Published
- 2016
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- View/download PDF
35. Myeloid conditional deletion and transgenic models reveal a threshold for the neutrophil survival factor Serpinb1.
- Author
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Burgener SS, Baumann M, Basilico P, Remold-O'Donnell E, Touw IP, and Benarafa C
- Subjects
- Alleles, Animals, Cell Survival, Gene Knock-In Techniques, Humans, Mice, Transgenic, Recombination, Genetic, Gene Deletion, Myeloid Cells metabolism, Neutrophils cytology, Serpins deficiency, Serpins genetics
- Abstract
Serpinb1 is an inhibitor of neutrophil granule serine proteases cathepsin G, proteinase-3 and elastase. One of its core physiological functions is to protect neutrophils from granule protease-mediated cell death. Mice lacking Serpinb1a (Sb1a-/-), its mouse ortholog, have reduced bone marrow neutrophil numbers due to cell death mediated by cathepsin G and the mice show increased susceptibility to lung infections. Here, we show that conditional deletion of Serpinb1a using the Lyz2-cre and Cebpa-cre knock-in mice effectively leads to recombination-mediated deletion in neutrophils but protein-null neutrophils were only obtained using the latter recombinase-expressing strain. Absence of Serpinb1a protein in neutrophils caused neutropenia and increased granule permeabilization-induced cell death. We then generated transgenic mice expressing human Serpinb1 in neutrophils under the human MRP8 (S100A8) promoter. Serpinb1a expression levels in founder lines correlated positively with increased neutrophil survival when crossed with Sb1a-/- mice, which had their defective neutrophil phenotype rescued in the higher expressing transgenic line. Using new conditional and transgenic mouse models, our study demonstrates the presence of a relatively low Serpinb1a protein threshold in neutrophils that is required for sustained survival. These models will also be helpful in delineating recently described functions of Serpinb1 in metabolism and cancer. more...
- Published
- 2016
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- View/download PDF
36. Small angle x-ray scattering with edge-illumination.
- Author
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Modregger P, Cremona TP, Benarafa C, Schittny JC, Olivo A, and Endrizzi M
- Subjects
- Animals, Contrast Media, Humans, Mice, Mice, 129 Strain, Scattering, Small Angle, X-Ray Diffraction, X-Rays, Image Processing, Computer-Assisted methods, Pulmonary Emphysema diagnosis, Radiography
- Abstract
Sensitivity to sub-pixel sample features has been demonstrated as a valuable capability of phase contrast x-ray imaging. Here, we report on a method to obtain angular-resolved small angle x-ray scattering distributions with edge-illumination- based imaging utilizing incoherent illumination from an x-ray tube. Our approach provides both the three established image modalities (absorption, differential phase and scatter strength), plus a number of additional contrasts related to unresolved sample features. The complementarity of these contrasts is experimentally validated by using different materials in powder form. As a significant application example we show that the extended complementary contrasts could allow the diagnosis of pulmonary emphysema in a murine model. In support of this, we demonstrate that the properties of the retrieved scattering distributions are consistent with the expectation of increased feature sizes related to pulmonary emphysema. Combined with the simplicity of implementation of edge-illumination, these findings suggest a high potential for exploiting extended sub-pixel contrasts in the diagnosis of lung diseases and beyond. more...
- Published
- 2016
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- View/download PDF
37. Increased Myeloid Cell Production and Lung Bacterial Clearance in Mice Exposed to Cigarette Smoke.
- Author
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Basilico P, Cremona TP, Oevermann A, Piersigilli A, and Benarafa C
- Subjects
- Animals, Bacterial Load, Disease Models, Animal, Granulocyte-Macrophage Colony-Stimulating Factor immunology, Host-Pathogen Interactions, Inflammation Mediators immunology, Interleukin-1beta immunology, Kinetics, Labyrinthitis immunology, Labyrinthitis microbiology, Lung microbiology, Mice, 129 Strain, Mice, Inbred C57BL, Monocytes immunology, Monocytes microbiology, Myeloid Cells microbiology, Neutrophils immunology, Neutrophils microbiology, Pneumonia, Pneumococcal microbiology, Pseudomonas Infections microbiology, Pseudomonas aeruginosa pathogenicity, Receptors, Interleukin-1 Type I genetics, Receptors, Interleukin-1 Type I metabolism, Serpins genetics, Serpins metabolism, Smoking adverse effects, Streptococcus pneumoniae pathogenicity, Immunity, Innate, Lung immunology, Myeloid Cells immunology, Pneumonia, Pneumococcal immunology, Pseudomonas Infections immunology, Pseudomonas aeruginosa immunology, Smoking immunology, Streptococcus pneumoniae immunology
- Abstract
Pneumonia is a leading cause of hospitalization in patients with chronic obstructive pulmonary disease (COPD). Although most patients with COPD are smokers, the effects of cigarette smoke exposure on clearance of lung bacterial pathogens and on immune and inflammatory responses are incompletely defined. Here, clearance of Streptococcus pneumoniae and Pseudomonas aeruginosa and associated immune responses were examined in mice exposed to cigarette smoke or after smoking cessation. Mice exposed to cigarette smoke for 6 weeks or 4 months demonstrated decreased lung bacterial burden compared with air-exposed mice when infected 16 to 24 hours after exposure. When infection was performed after smoke cessation, bacterial clearance kinetics of mice previously exposed to smoke reversed to levels comparable to those of control mice, suggesting that the observed defects were not dependent on adaptive immunological memory to bacterial determinants found in smoke. Comparing cytokine levels and myeloid cell production before infection in mice exposed to cigarette smoke with mice never exposed or after smoke cessation revealed that reduced bacterial burden was most strongly associated with higher levels of IL-1β and granulocyte-macrophage colony-stimulating factor in the lungs and with increased neutrophil reserve and monocyte turnover in the bone marrow. Using Serpinb1a-deficient mice with reduced neutrophil numbers and treatment with granulocyte colony-stimulating factor showed that increased neutrophil numbers contribute only in part to the effect of smoke on infection. Our findings indicate that cigarette smoke induces a temporary and reversible increase in clearance of lung pathogens, which correlates with local inflammation and increased myeloid cell output from the bone marrow. more...
- Published
- 2016
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- View/download PDF
38. CXCR1 Regulates Pulmonary Anti-Pseudomonas Host Defense.
- Author
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Carevic M, Öz H, Fuchs K, Laval J, Schroth C, Frey N, Hector A, Bilich T, Haug M, Schmidt A, Autenrieth SE, Bucher K, Beer-Hammer S, Gaggar A, Kneilling M, Benarafa C, Gao JL, Murphy PM, Schwarz S, Moepps B, and Hartl D more...
- Subjects
- Animals, Cells, Cultured, Disease Models, Animal, Female, Host-Pathogen Interactions, Humans, Immunity, Innate, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Reactive Oxygen Species metabolism, Receptors, Interleukin-8A immunology, Respiratory Mucosa microbiology, Toll-Like Receptor 5 genetics, Toll-Like Receptor 5 metabolism, Young Adult, Cystic Fibrosis immunology, Neutrophils immunology, Pseudomonas Infections immunology, Pseudomonas aeruginosa immunology, Pulmonary Disease, Chronic Obstructive immunology, Receptors, Interleukin-8A metabolism, Respiratory Mucosa immunology
- Abstract
Pseudomonas aeruginosa is a key opportunistic pathogen causing disease in cystic fibrosis (CF) and other lung diseases such as chronic obstructive pulmonary disease (COPD). However, the pulmonary host defense mechanisms regulating anti-P. aeruginosa immunity remain incompletely understood. Here we demonstrate, by studying an airway P. aeruginosa infection model, in vivo bioluminescence imaging, neutrophil effector responses and human airway samples, that the chemokine receptor CXCR1 regulates pulmonary host defense against P. aeruginosa. Mechanistically, CXCR1 regulates anti-Pseudomonas neutrophil responses through modulation of reactive oxygen species and interference with Toll-like receptor 5 expression. These studies define CXCR1 as a novel, noncanonical chemokine receptor that regulates pulmonary anti-Pseudomonas host defense with broad implications for CF, COPD and other infectious lung diseases., (© 2016 S. Karger AG, Basel.) more...
- Published
- 2016
- Full Text
- View/download PDF
39. CXCL14: the Swiss army knife chemokine.
- Author
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Benarafa C and Wolf M
- Subjects
- Animals, Chemokines, CXC genetics, Humans, Lung microbiology, Mice, Neoplasms therapy, Anti-Bacterial Agents immunology, Chemokines, CXC immunology, Gram-Negative Bacteria immunology, Inflammation immunology, Lung immunology, Neoplasms immunology
- Published
- 2015
- Full Text
- View/download PDF
40. Tumor-induced inflammation alters neutrophil phenotype and disease progression.
- Author
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Benarafa C
- Subjects
- Breast Neoplasms pathology, Breast Neoplasms therapy, Cytokines physiology, Disease Progression, Female, Humans, Immunosuppression Therapy, Neutrophil Infiltration, Phenotype, Breast Neoplasms immunology, Neutrophils immunology
- Abstract
Neutrophils are essential to combat infectious agents but contribute to collateral inflammatory damage. Likewise, neutrophils can kill cancer cells and have been shown to promote malignant growth and metastasis through immunosuppressive functions. Two articles in a recent issue of Nature reveal new mechanisms by which tumors induce changes in neutrophil phenotype through production of inflammatory cytokines. Although the two studies report different outcomes on the effects of neutrophils on tumor growth and metastasis, they delineate novel molecular pathways influencing neutrophil phenotype that may provide new approaches to harnessing neutrophil functions in the treatment of cancer. more...
- Published
- 2015
- Full Text
- View/download PDF
41. CXCL14 displays antimicrobial activity against respiratory tract bacteria and contributes to clearance of Streptococcus pneumoniae pulmonary infection.
- Author
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Dai C, Basilico P, Cremona TP, Collins P, Moser B, Benarafa C, and Wolf M
- Subjects
- Adenosine Monophosphate metabolism, Amino Acid Sequence, Animals, Anti-Infective Agents chemistry, Cell Membrane drug effects, Cell Membrane metabolism, Chemokines, CXC chemistry, Chemokines, CXC genetics, Chemokines, CXC metabolism, Chemotaxis drug effects, DNA, Bacterial, Disease Models, Animal, Interleukin-8 pharmacology, Lung immunology, Lung metabolism, Lung microbiology, Lung pathology, Mice, Mice, Knockout, Microbial Sensitivity Tests, Models, Molecular, Myeloblastin metabolism, Peptide Fragments chemistry, Peptide Fragments pharmacology, Permeability drug effects, Pneumococcal Infections drug therapy, Pneumococcal Infections genetics, Protein Binding, Protein Conformation, Protein Interaction Domains and Motifs, Proteolysis, Respiratory Tract Infections drug therapy, Respiratory Tract Infections genetics, Streptococcus pneumoniae ultrastructure, Anti-Infective Agents pharmacology, Chemokines, CXC pharmacology, Pneumococcal Infections immunology, Respiratory Tract Infections immunology, Streptococcus pneumoniae drug effects, Streptococcus pneumoniae immunology
- Abstract
CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia., (Copyright © 2015 by The American Association of Immunologists, Inc.) more...
- Published
- 2015
- Full Text
- View/download PDF
42. Neutrophils: Between host defence, immune modulation, and tissue injury.
- Author
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Kruger P, Saffarzadeh M, Weber AN, Rieber N, Radsak M, von Bernuth H, Benarafa C, Roos D, Skokowa J, and Hartl D
- Subjects
- Animals, Cell Survival immunology, Humans, Infections pathology, Inflammation immunology, Inflammation pathology, Neutrophils pathology, Cell Movement immunology, Extracellular Traps immunology, Infections immunology, Neutrophils immunology
- Abstract
Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage. more...
- Published
- 2015
- Full Text
- View/download PDF
43. The generation of neutrophils in the bone marrow is controlled by autophagy.
- Author
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Rožman S, Yousefi S, Oberson K, Kaufmann T, Benarafa C, and Simon HU
- Subjects
- Animals, Cell Differentiation physiology, Inbreeding, Mice, Mice, Inbred C57BL, Mice, Transgenic, Neutrophils metabolism, Signal Transduction, Autophagy physiology, Bone Marrow Cells cytology, Neutrophils cytology
- Abstract
Autophagy has been demonstrated to have an essential function in several cellular hematopoietic differentiation processes, for example, the differentiation of reticulocytes. To investigate the role of autophagy in neutrophil granulopoiesis, we studied neutrophils lacking autophagy-related (Atg) 5, a gene encoding a protein essential for autophagosome formation. Using Cre-recombinase mediated gene deletion, Atg5-deficient neutrophils showed no evidence of abnormalities in morphology, granule protein content, apoptosis regulation, migration, or effector functions. In such mice, however, we observed an increased proliferation rate in the neutrophil precursor cells of the bone marrow as well as an accelerated process of neutrophil differentiation, resulting in an accumulation of mature neutrophils in the bone marrow, blood, spleen, and lymph nodes. To directly study the role of autophagy in neutrophils, we employed an in vitro model of differentiating neutrophils that allowed modulating the levels of ATG5 expression, or, alternatively, intervening pharmacologically with autophagy-regulating drugs. We could show that autophagic activity correlated inversely with the rate of neutrophil differentiation. Moreover, pharmacological inhibition of p38 MAPK or mTORC1 induced autophagy in neutrophilic precursor cells and blocked their differentiation, suggesting that autophagy is negatively controlled by the p38 MAPK-mTORC1 signaling pathway. On the other hand, we obtained no evidence for an involvement of the PI3K-AKT or ERK1/2 signaling pathways in the regulation of neutrophil differentiation. Taken together, these findings show that, in contrast to erythropoiesis, autophagy is not essential for neutrophil granulopoiesis, having instead a negative impact on the generation of neutrophils. Thus, autophagy and differentiation exhibit a reciprocal regulation by the p38-mTORC1 axis. more...
- Published
- 2015
- Full Text
- View/download PDF
44. DAPK2 positively regulates motility of neutrophils and eosinophils in response to intermediary chemoattractants.
- Author
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Geering B, Stoeckle C, Rozman S, Oberson K, Benarafa C, and Simon HU
- Subjects
- Animals, Cell Adhesion drug effects, Cell Polarity drug effects, Cell Survival drug effects, Death-Associated Protein Kinases antagonists & inhibitors, Disease Models, Animal, Eosinophils drug effects, Eosinophils enzymology, Humans, Inflammation pathology, Jurkat Cells, Mice, Myosin Light Chains metabolism, Neutrophils drug effects, Peritonitis pathology, Phosphorylation, Protein Kinase Inhibitors pharmacology, Small Molecule Libraries pharmacology, Cell Movement drug effects, Chemotactic Factors pharmacology, Death-Associated Protein Kinases metabolism, Eosinophils cytology, Neutrophils cytology, Neutrophils enzymology
- Abstract
The tight regulation of granulocyte chemotaxis is crucial for initiation and resolution of inflammation. Here, we show that DAPK2, a Ca(2+)/CaM-sensitive serine/threonine kinase known to modulate cell death in various cell types, is a novel regulator of migration in granulocytes. We demonstrate that human neutrophils and eosinophils express DAPK2 but unlike other leukocytes, no DAPK1 or DAPK3 protein. When DAPK activities were blocked by inhibitors, we found that neither granulocyte lifespan nor phagocytosis was affected. However, such pharmacological inactivation of DAPK activity abolished motility of granulocytes in response to intermediary but not end-target chemoattractants ex vivo. The defect in chemotaxis in DAPK2-inactive granulocytes is likely a result of reduced polarization of the cells, mediated by a lack of MLC phosphorylation, resulting in radial F-actin and pseudopod formation. As neutrophils treated with DAPKi also showed reduced recruitment to the site of inflammation in a mouse peritonitis model, DAPK2 may be a novel target for anti-inflammatory therapies. more...
- Published
- 2014
- Full Text
- View/download PDF
45. SerpinB1 deficiency is not associated with increased susceptibility to pulmonary emphysema in mice.
- Author
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Cremona TP, Tschanz SA, von Garnier C, and Benarafa C
- Subjects
- Animals, Bronchoalveolar Lavage Fluid cytology, Disease Models, Animal, Disease Susceptibility metabolism, Disease Susceptibility pathology, Mice, Mice, 129 Strain, Mice, Knockout, Mutation genetics, Pneumonia chemically induced, Pneumonia genetics, Pneumonia pathology, Serpins deficiency, Smoke adverse effects, Pulmonary Emphysema genetics, Pulmonary Emphysema metabolism, Pulmonary Emphysema pathology, Serpins metabolism
- Abstract
Chronic obstructive pulmonary disease (COPD) is characterized by emphysema and chronic bronchitis and is a leading cause of morbidity and mortality worldwide. Tobacco smoke and deficiency in α1-antitrypsin (AAT) are the most prominent environmental and genetic risk factors, respectively. Yet the pathogenesis of COPD is not completely elucidated. Disease progression appears to include a vicious circle driven by self-perpetuating lung inflammation, endothelial and epithelial cell death, and proteolytic degradation of extracellular matrix proteins. Like AAT, serpinB1 is a potent inhibitor of serine proteases including neutrophil elastase and cathepsin G. Because serpinB1 is expressed in myeloid and lung epithelial cells and is protective during lung infections, we investigated the role of serpinB1 in preventing age-related and cigarette smoke-induced emphysema in mice. Fifteen-month-old mice showed increased lung volume and decreased pulmonary function compared with young adult mice (3 mo old), but no differences were observed between serpinB1-deficient (KO) and wild-type (WT) mice. Chronic exposure to secondhand cigarette smoke resulted in structural emphysematous changes compared with respective control mice, but no difference in lung morphometry was observed between genotypes. Of note, the different pattern of stereological changes induced by age and cigarette smoke suggest distinct mechanisms leading to increased airway volume. Finally, expression of intracellular and extracellular protease inhibitors were differently regulated in lungs of WT and KO mice following smoke exposure; however, activity of proteases was not significantly altered. In conclusion, we showed that, although AAT and serpinB1 are similarly potent inhibitors of neutrophil proteases, serpinB1 deficiency is not associated with more severe emphysema. more...
- Published
- 2013
- Full Text
- View/download PDF
46. SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G.
- Author
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Baumann M, Pham CT, and Benarafa C
- Subjects
- Animals, Cathepsin G metabolism, Cell Survival genetics, Cells, Cultured, Down-Regulation genetics, Female, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Neutrophils metabolism, Organ Specificity genetics, Serpins genetics, Serpins metabolism, Cathepsin G antagonists & inhibitors, Neutrophils physiology, Serpins physiology
- Abstract
Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1(-/-)) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia of sB1(-/-) mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1(-/-) mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1(-/-) PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent l-leucyl-l-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity. more...
- Published
- 2013
- Full Text
- View/download PDF
47. Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice.
- Author
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Stolley JM, Gong D, Farley K, Zhao P, Cooley J, Crouch EC, Benarafa C, and Remold-O'Donnell E
- Subjects
- Animals, Bronchoalveolar Lavage Fluid, Cathepsin G metabolism, Female, Lung Injury immunology, Lung Injury metabolism, Lung Injury microbiology, Mice, Mice, 129 Strain, Mice, Knockout, Myeloblastin metabolism, Neutrophils enzymology, Pancreatic Elastase metabolism, Pneumonia, Bacterial immunology, Pneumonia, Bacterial metabolism, Pneumonia, Bacterial microbiology, Pseudomonas Infections immunology, Pseudomonas Infections metabolism, Pseudomonas Infections microbiology, Pseudomonas aeruginosa immunology, Pulmonary Surfactant-Associated Protein D genetics, Serpins deficiency, Pulmonary Surfactant-Associated Protein D metabolism, Serpins genetics
- Abstract
Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis. more...
- Published
- 2012
- Full Text
- View/download PDF
48. Critical role of serpinB1 in regulating inflammatory responses in pulmonary influenza infection.
- Author
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Gong D, Farley K, White M, Hartshorn KL, Benarafa C, and Remold-O'Donnell E
- Subjects
- Animals, Cell Death, Cytokines genetics, Cytokines metabolism, Gene Expression Regulation physiology, Inflammation pathology, Influenza A Virus, H3N2 Subtype, Lung Diseases pathology, Mice, Mice, Knockout, Orthomyxoviridae Infections immunology, Serpins genetics, Time Factors, Inflammation metabolism, Lung pathology, Lung Diseases virology, Orthomyxoviridae Infections pathology, Serpins metabolism
- Abstract
Background: Excessive inflammatory host response increases morbidity and mortality associated with seasonal respiratory influenza, and highly pathogenic virus strains are characterized by massive infiltration of monocytes and/or macrophages that produce a storm of injurious cytokines., Methods: Here, we examined the role in respiratory influenza of serpinB1, an endogenous inhibitor of the serine proteases elastase, cathepsin G, and proteinase-3, increasingly recognized as regulators of inflammation., Results: After challenge with high-dose surfactant protein-D (SP-D)-sensitive influenza A/Philadelphia/82 (H3N2), serpinB1(-/-) mice died earlier and in greater numbers than did wild-type mice. Sublethally infected animals suffered increased morbidity, delayed resolution of epithelial injury, and increased immune cell death. Viral clearance and SP-D/SP-A upregulation were unimpaired and so were early virus-induced cytokine and chemokine burst and influx of large numbers of neutrophils and monocytes. Whereas initial cytokines and chemokines rapidly cleared in wild-type mice, TNF-α, IL-6, KC/CXCL1, G-CSF, IL-17A, and MCP-1/CCL2 remained elevated in serpinB1(-/-) mice. Monocyte-derived cells were the dominant immune cells in influenza-infected lungs, and those from serpinB1(-/-) mice produced excessive IL-6 and TNF-α when tested ex vivo. Pulmonary γδ T-cells that produced IL-17A were also increased., Conclusions: Because viral clearance was unimpaired, the study highlights the critical role of serpinB1 in mitigating inflammation and restricting pro-inflammatory cytokine production in influenza infection. more...
- Published
- 2011
- Full Text
- View/download PDF
49. SerpinB1 protects the mature neutrophil reserve in the bone marrow.
- Author
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Benarafa C, LeCuyer TE, Baumann M, Stolley JM, Cremona TP, and Remold-O'Donnell E
- Subjects
- Animals, Blotting, Western, Bone Marrow Cells cytology, Bone Marrow Cells metabolism, Cell Differentiation, Cell Separation, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Granulocyte Colony-Stimulating Factor immunology, Granulocyte Colony-Stimulating Factor metabolism, Humans, Mice, Mice, Knockout, Neutrophils cytology, Neutrophils metabolism, Reverse Transcriptase Polymerase Chain Reaction, Serine Proteases immunology, Serine Proteases metabolism, Serpins metabolism, Bone Marrow Cells immunology, Neutrophils immunology, Serpins immunology
- Abstract
SerpinB1 is among the most efficient inhibitors of neutrophil serine proteases--NE, CG, and PR-3--and we investigated here its role in neutrophil development and homeostasis. We found that serpinB1 is expressed in all human bone marrow leukocytes, including stem and progenitor cells. Expression levels were highest in the neutrophil lineage and peaked at the promyelocyte stage, coincident with the production and packaging of the target proteases. Neutrophil numbers were decreased substantially in the bone marrow of serpinB1(-/-) mice. This cellular deficit was associated with an increase in serum G-CSF levels. On induction of acute pulmonary injury, neutrophils were recruited to the lungs, causing the bone marrow reserve pool to be completely exhausted in serpinB1(-/-) mice. Numbers of myeloid progenitors were normal in serpinB1(-/-) bone marrow, coincident with the absence of target protease expression at these developmental stages. Maturation arrest of serpinB1(-/-) neutrophils was excluded by the normal CFU-G growth in vitro and the normal expression in mature neutrophils of early and late differentiation markers. Normal absolute numbers of proliferating neutrophils and pulse-chase kinetic studies in vivo showed that the bone marrow deficit in serpinB1(-/-) mice was largely restricted to mature, postmitotic neutrophils. Finally, upon overnight culture, apoptosis and necrosis were greater in purified bone marrow neutrophils from serpinB1(-/-) compared with WT mice. Collectively, these findings demonstrate that serpinB1 sustains a healthy neutrophil reserve that is required in acute immune responses. more...
- Published
- 2011
- Full Text
- View/download PDF
50. DNase 2 is the main DNA-degrading enzyme of the stratum corneum.
- Author
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Fischer H, Scherz J, Szabo S, Mildner M, Benarafa C, Torriglia A, Tschachler E, and Eckhart L
- Subjects
- Animals, Humans, Mice, Serpins metabolism, Skin, Artificial, DNA metabolism, Endodeoxyribonucleases metabolism, Epidermis enzymology
- Abstract
The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA. more...
- Published
- 2011
- Full Text
- View/download PDF
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