Neutrophil-Specific Knockdown of β2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis

β2-integrins are heterodimeric surface receptors that are expressed specifically by leukocytes and consist of a variable α (CD11a-d) and a common β-subunit (CD18). Functional impairment of CD18, which causes leukocyte adhesion deficiency type-1 results in an immunocompromised state characterized by severe infections, such as invasive pulmonary aspergillosis (IPA). The underlying immune defects have largely been attributed to an impaired migratory and phagocytic activity of polymorphonuclear granulocytes (PMN). However, the exact contribution of β2-integrins for PMN functions in-vivo has not been elucidated yet, since the mouse models available so far display a constitutive CD18 knockout (CD18-/- or CD18hypo). To determine the PMN-specific role of β2-integrins for innate effector functions and pathogen control, we generated a mouse line with a Ly6G-specific knockdown of the common β-subunit (CD18Ly6G cKO). We characterized CD18Ly6G cKO mice in-vitro to confirm the PMN-specific knockdown of β2-integrins. Next, we investigated the clinical course of IPA in A. fumigatus infected CD18Ly6G cKO mice with regard to the fungal burden, pulmonary inflammation and PMN response towards A. fumigatus. Our results revealed that the β2-integrin knockdown was restricted to PMN and that CD18Ly6G cKO mice showed an aggravated course of IPA. In accordance, we observed a higher fungal burden and lower levels of proinflammatory innate cytokines, such as TNF-α, in lungs of IPA-infected CD18Ly6G cKO mice. Bronchoalveolar lavage revealed higher levels of CXCL1, a stronger PMN-infiltration, but concomitantly elevated apoptosis of PMN in lungs of CD18Ly6G cKO mice. Ex-vivo analysis further unveiled a strong impairment of PMN effector function, as reflected by an attenuated phagocytic activity, and a diminished generation of reactive oxygen species (ROS) and neutrophil-extracellular traps (NET) in CD18-deficient PMN. Overall, our study demonstrates that β2-integrins are required specifically for PMN effector functions and contribute to the clearance of A. fumigatus by infiltrating PMN, and the establishment of an inflammatory microenvironment in infected lungs.

[1]  M. Bros,et al.  β2 Integrins—Multi-Functional Leukocyte Receptors in Health and Disease , 2020, International journal of molecular sciences.

[2]  K. Midwood,et al.  More Than Just Attractive: How CCL2 Influences Myeloid Cell Behavior Beyond Chemotaxis , 2019, Front. Immunol..

[3]  T. K. van den Berg,et al.  Activated neutrophils exert myeloid-derived suppressor cell activity damaging T cells beyond repair. , 2019, Blood advances.

[4]  Jaewoo Hong,et al.  Neutrophilic Granule Protein Is a Novel Murine LPS Antagonist , 2019, bioRxiv.

[5]  R. Alon,et al.  Distinct Compartmentalization of the Chemokines CXCL1 and CXCL2 and the Atypical Receptor ACKR1 Determine Discrete Stages of Neutrophil Diapedesis , 2018, Immunity.

[6]  M. Bros,et al.  CD11b Regulates Fungal Outgrowth but Not Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis , 2018, Front. Immunol..

[7]  M. Gunzer,et al.  Differential attenuation of β2 integrin-dependent and -independent neutrophil migration by Ly6G ligation , 2018, bioRxiv.

[8]  E. Podrez,et al.  Oxidative modifications of extracellular matrix promote the second wave of inflammation via β2 integrins. , 2018, Blood.

[9]  E. Pearlman,et al.  Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and β-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3 , 2018, Front. Immunol..

[10]  G. Braus,et al.  Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death , 2017, Science.

[11]  H. Schild,et al.  ADAMTS-13 regulates neutrophil recruitment in a mouse model of invasive pulmonary aspergillosis , 2017, Scientific Reports.

[12]  Chengcai Lai,et al.  C-C Motif Chemokine Ligand 2 (CCL2) Mediates Acute Lung Injury Induced by Lethal Influenza H7N9 Virus , 2017, Front. Microbiol..

[13]  Xiaodong Zhang,et al.  The Diverse Biological Functions of Neutrophils, Beyond the Defense Against Infections , 2017, Inflammation.

[14]  Jun R Huh,et al.  Central Role of IL-23 and IL-17 Producing Eosinophils as Immunomodulatory Effector Cells in Acute Pulmonary Aspergillosis and Allergic Asthma , 2017, PLoS pathogens.

[15]  Xiaodong Zhang,et al.  The Diverse Biological Functions of Neutrophils, Beyond the Defense Against Infections , 2016, Inflammation.

[16]  K. Ley,et al.  Biology and structure of leukocyte β 2 integrins and their role in inflammation , 2016, F1000Research.

[17]  J. Reichner,et al.  Neutrophil Integrins and Matrix Ligands and NET Release , 2016, Front. Immunol..

[18]  F. Lamoth.  Galactomannan and 1,3-β-d-Glucan Testing for the Diagnosis of Invasive Aspergillosis , 2016, Journal of fungi.

[19]  J. Cavaillon,et al.  Reducing hypoxia and inflammation during invasive pulmonary aspergillosis by targeting the Interleukin-1 receptor , 2016, Scientific Reports.

[20]  H. Thorlacius,et al.  Platelet‐Derived CCL5 Regulates CXC Chemokine Formation and Neutrophil Recruitment in Acute Experimental Colitis , 2016, Journal of cellular physiology.

[21]  T. K. van den Berg,et al.  Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects , 2016, The Journal of Immunology.

[22]  F. de Chaumont,et al.  Absence of Fungal Spore Internalization by Bronchial Epithelium in Mouse Models Evidenced by a New Bioimaging Approach and Transmission Electronic Microscopy. , 2015, The American journal of pathology.

[23]  A. Waisman,et al.  Catchup: a mouse model for imaging-based tracking and modulation of neutrophil granulocytes , 2015, Nature Methods.

[24]  Steven G. E. Braem,et al.  Effective Neutrophil Phagocytosis of Aspergillus fumigatus Is Mediated by Classical Pathway Complement Activation , 2015, Journal of Innate Immunity.

[25]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[26]  H. Einsele,et al.  Human dendritic cell subsets display distinct interactions with the pathogenic mould Aspergillus fumigatus. , 2014, International journal of medical microbiology : IJMM.

[27]  Michael Poidinger,et al.  High-dimensional analysis of the murine myeloid cell system , 2014, Nature Immunology.

[28]  H. Schild,et al.  Oxidative burst and neutrophil elastase contribute to clearance of Aspergillus fumigatus pneumonia in mice. , 2014, Immunobiology.

[29]  P. Taylor,et al.  Integrin CD11b positively regulates TLR4-induced signalling pathways in dendritic cells but not in macrophages , 2014, Nature Communications.

[30]  C. Steele,et al.  Eosinophil Deficiency Compromises Lung Defense against Aspergillus fumigatus , 2013, Infection and Immunity.

[31]  Szabina Fodor,et al.  Neutrophil cell surface receptors and their intracellular signal transduction pathways , 2013, International immunopharmacology.

[32]  E. Parisini,et al.  Ly6 family proteins in neutrophil biology , 2013, Journal of leukocyte biology.

[33]  M. Netea,et al.  Hidden Killers: Human Fungal Infections , 2012, Science Translational Medicine.

[34]  A. Weyrich,et al.  Lessons from rare maladies: leukocyte adhesion deficiency syndromes , 2012, Current opinion in hematology.

[35]  N. Hotchin,et al.  Signalling mechanisms of the leukocyte integrin αMβ2: Current and future perspectives , 2012, Biology of the cell.

[36]  P. Nigrovic,et al.  Ly6G ligation blocks recruitment of neutrophils via a β2-integrin-dependent mechanism. , 2012, Blood.

[37]  Michelle Momany,et al.  Fungal antioxidant pathways promote survival against neutrophils during infection. , 2012, The Journal of clinical investigation.

[38]  M. Burdick,et al.  Neutrophils Mediate Maturation and Efflux of Lung Dendritic Cells in Response to Aspergillus fumigatus Germ Tubes , 2012, Infection and Immunity.

[39]  P. Taylor,et al.  Class IA Phosphoinositide 3-Kinase β and δ Regulate Neutrophil Oxidase Activation in Response to Aspergillus fumigatus Hyphae , 2011, The Journal of Immunology.

[40]  W. Fonzi,et al.  Regulation of Innate Immune Response to Candida albicans Infections by αMβ2-Pra1p Interaction , 2011, Infection and Immunity.

[41]  C. Garlanda,et al.  Role of complement and Fc{gamma} receptors in the protective activity of the long pentraxin PTX3 against Aspergillus fumigatus. , 2010, Blood.

[42]  Vandana Saini,et al.  MCP-1: chemoattractant with a role beyond immunity: a review. , 2010, Clinica chimica acta; international journal of clinical chemistry.

[43]  Zhen-yu Huang,et al.  Interaction of Two Phagocytic Host Defense Systems , 2010, The Journal of Biological Chemistry.

[44]  E. Anaissie,et al.  Factors associated with mortality in transplant patients with invasive aspergillosis. , 2010, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[45]  D. Villines,et al.  S100A8 and S100A9 inhibit neutrophil oxidative metabolism in-vitro: Involvement of adenosine metabolites , 2010, Free radical research.

[46]  K. Clément,et al.  CCL5 Promotes Macrophage Recruitment and Survival in Human Adipose Tissue , 2010, Arteriosclerosis, thrombosis, and vascular biology.

[47]  S. Frøland,et al.  Activation of Platelets by Aspergillus fumigatus and Potential Role of Platelets in the Immunopathogenesis of Aspergillosis , 2009, Infection and Immunity.

[48]  T. Kuijpers,et al.  Complement receptor 3, not Dectin-1, is the major receptor on human neutrophils for beta-glucan-bearing particles. , 2009, Molecular immunology.

[49]  C. Lowell,et al.  Neutrophil-specific deletion of Syk kinase results in reduced host defense to bacterial infection. , 2009, Blood.

[50]  S. Zahler,et al.  Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators , 2009, Arteriosclerosis, thrombosis, and vascular biology.

[51]  Matthew J. Craig,et al.  CCL2 and Interleukin-6 Promote Survival of Human CD11b+ Peripheral Blood Mononuclear Cells and Induce M2-type Macrophage Polarization* , 2009, The Journal of Biological Chemistry.

[52]  W. Nacken,et al.  Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans , 2009, PLoS pathogens.

[53]  T. Hohl,et al.  Essential role for neutrophils but not alveolar macrophages at early time points following Aspergillus fumigatus infection. , 2009, The Journal of infectious diseases.

[54]  H. Langer,et al.  Leukocyte – endothelial interactions in inflammation , 2009, Journal of cellular and molecular medicine.

[55]  R. Johns,et al.  Pulmonary epithelium is a prominent source of proteinase-activated receptor-1-inducible CCL2 in pulmonary fibrosis. , 2009, American journal of respiratory and critical care medicine.

[56]  C. Cordonnier,et al.  Phagocytosis of Aspergillus fumigatus conidia by primary nasal epithelial cells in vitro , 2008, BMC Microbiology.

[57]  H. Fuller,et al.  Prevalence of culturable airborne spores of selected allergenic and pathogenic fungi in outdoor air , 2008 .

[58]  T. Welte,et al.  Lung-specific overexpression of CC chemokine ligand (CCL) 2 enhances the host defense to Streptococcus pneumoniae infection in mice: role of the CCL2-CCR2 axis. , 2007, Journal of immunology.

[59]  Matt Post,et al.  Invasive fungal infections in allogeneic and autologous stem cell transplant recipients: a single‐center study of 166 transplanted patients , 2007, Transplant infectious disease : an official journal of the Transplantation Society.

[60]  T. Welte,et al.  Lung-Specific Overexpression of CC Chemokine Ligand (CCL) 2 Enhances the Host Defense to Streptococcus pneumoniae Infection in Mice: Role of the CCL2-CCR2 Axis1 , 2007, The Journal of Immunology.

[61]  J. Heesemann,et al.  Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin‐1 beta‐glucan receptor and Toll‐like receptor 2 , 2007, Cellular microbiology.

[62]  V. Wahn,et al.  Novel cell death program leads to neutrophil extracellular traps , 2007, The Journal of Cell Biology.

[63]  D. Vestweber,et al.  Active MAC-1 (CD11b/CD18) on DCs inhibits full T-cell activation. , 2007, Blood.

[64]  M. Feldmesser.  Role of Neutrophils in Invasive Aspergillosis▿ , 2006, Infection and Immunity.

[65]  J. Tregoning,et al.  Role of CCL5 (RANTES) in Viral Lung Disease , 2006, Journal of Virology.

[66]  W. Seeger,et al.  Alveolar Epithelial Cells Direct Monocyte Transepithelial Migration upon Influenza Virus Infection: Impact of Chemokines and Adhesion Molecules1 , 2006, The Journal of Immunology.

[67]  R. Grillot,et al.  Human Dendritic Cells following Aspergillus fumigatus Infection Express the CCR7 Receptor and a Differential Pattern of Interleukin-12 (IL-12), IL-23, and IL-27 Cytokines, Which Lead to a Th1 Response , 2006, Infection and Immunity.

[68]  T. van der Poll,et al.  CD11b limits bacterial outgrowth and dissemination during murine pneumococcal pneumonia. , 2005, The Journal of infectious diseases.

[69]  L. Truedsson,et al.  Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum , 2005, European journal of immunology.

[70]  E. Génot,et al.  Inhibitory control of TGF‐β1 on the activation of Rap1, CD11b, and transendothelial migration of leukocytes , 2005, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[71]  T. Geijtenbeek,et al.  Neutrophils mediate immune modulation of dendritic cells through glycosylation-dependent interactions between Mac-1 and DC-SIGN , 2005, The Journal of experimental medicine.

[72]  J. Jimenez-Heffernan,et al.  Dendritic Cell-Specific Intercellular Adhesion Molecule 3-Grabbing Nonintegrin Mediates Binding and Internalization of Aspergillus fumigatus Conidia by Dendritic Cells and Macrophages1 , 2004, The Journal of Immunology.

[73]  A. Issekutz,et al.  The CD11/CD18 (β2) integrins modulate neutrophil caspase activation and survival following TNF‐α or endotoxin induced transendothelial migration , 2004 .

[74]  J. Latgé,et al.  Aspergillus fumigatus antigens activate innate immune cells via toll‐like receptors 2 and 4 , 2004, British journal of haematology.

[75]  S. Han,et al.  Aggregation of β2 integrins activates human neutrophils through the IκB/NF‐κB pathway , 2004, Journal of leukocyte biology.

[76]  M. Wellner,et al.  Integrins and Cytokines Activate Nuclear Transcription Factor-κB in Human Neutrophils* , 2004, Journal of Biological Chemistry.

[77]  S. Kunkel.  Faculty Opinions recommendation of Cross-talk in the innate immune system: neutrophils instruct recruitment and activation of dendritic cells during microbial infection. , 2003 .

[78]  T. Curiel,et al.  Cross-Talk in the Innate Immune System: Neutrophils Instruct Recruitment and Activation of Dendritic Cells during Microbial Infection 1 , 2003, The Journal of Immunology.

[79]  Bin Zhang,et al.  Elucidation of Molecular Events Leading to Neutrophil Apoptosis following Phagocytosis , 2003, Journal of Biological Chemistry.

[80]  W. Seeger,et al.  Monocytes Are Potent Facilitators of Alveolar Neutrophil Emigration During Lung Inflammation: Role of the CCL2-CCR2 Axis1 , 2003, The Journal of Immunology.

[81]  C. Ryan,et al.  CD18 Dependency of Transendothelial Neutrophil Migration Differs During Acute Pulmonary Inflammation1 , 2001, The Journal of Immunology.

[82]  J. Latgé.  The pathobiology of Aspergillus fumigatus. , 2001, Trends in microbiology.

[83]  Z. Sułowska,et al.  Spontaneous Apoptosis of Neutrophils in Whole Blood and its Relation to Apoptosis Gene Proteins , 2000, Scandinavian journal of immunology.

[84]  S. M. Propst,et al.  Proinflammatory and Th2-Derived Cytokines Modulate CD40-Mediated Expression of Inflammatory Mediators in Airway Epithelia: Implications for the Role of Epithelial CD40 in Airway Inflammation , 2000, The Journal of Immunology.

[85]  L. Dekker,et al.  Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils. , 2000, The Biochemical journal.

[86]  M. Ehlers,et al.  CR3: a general purpose adhesion-recognition receptor essential for innate immunity. , 2000, Microbes and infection.

[87]  Z. Ding,et al.  Relative contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration. , 1999, Journal of immunology.

[88]  M. Scott,et al.  Effects of CD18 deficiency on the emigration of murine neutrophils during pneumonia. , 1999, Journal of immunology.

[89]  J. Schröder,et al.  Th1- and Th2-type cytokines regulate the expression and production of eotaxin and RANTES by human lung fibroblasts. , 1999, American journal of respiratory cell and molecular biology.

[90]  P. Gaehtgens,et al.  β2 integrins (CD11/CD18) promote apoptosis of human neutrophils , 1997, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[91]  S Askari,et al.  A novel role for the beta 2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. , 1996, Immunity.

[92]  D. Graves,et al.  Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes. , 1992, Journal of immunology.

[93]  B. Rollins,et al.  Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes. , 1991, Blood.

[94]  F. Parhami,et al.  Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[95]  T. Springer,et al.  The severe and moderate phenotypes of heritable Mac-1, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features. , 1985, The Journal of infectious diseases.

[96]  B. Strom,et al.  Prolonged granulocytopenia: the major risk factor for invasive pulmonary aspergillosis in patients with acute leukemia. , 1984, Annals of internal medicine.

[97]  R. Clark,et al.  Damage to Aspergillus fumigatus and Rhizopus oryzae Hyphae by Oxidative and Nonoxidative Microbicidal Products of Human Neutrophils In Vitro , 1982, Infection and immunity.

[98]  H. Malech,et al.  Fungal infection in chronic granulomatous disease. The importance of the phagocyte in defense against fungi. , 1981, The American journal of medicine.

[99]  A. Etzioni.  Leukocyte Adhesion Deficiency Syndromes , 2020, Encyclopedia of Medical Immunology.

[100]  M. Herbst,et al.  PHAGOCYTES , GRANULOCYTES , AND MYELOPOIESIS Two independent killing mechanisms of Candida albicans by human neutrophils : evidence from innate immunity defects , 2014 .

[101]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[102]  T. Yoshimura,et al.  IFN-gamma-mediated survival enables human neutrophils to produce MCP-1/CCL2 in response to activation by TLR ligands. , 2007, Journal of immunology.

[103]  S. Heffelfinger,et al.  Leukocyte adhesion deficiency: clinical and postmortem observations. , 1992, Pediatric pathology.

[104]  C. d’Enfert,et al.  Human Fungal Infections. , 1951, British medical journal.

[105]  K. Matsushima,et al.  Brief Definitive Report Purification and Characterization of a Novel Monocyte Chemotactic and Activating Factor Produced by a Human Myelomonocytic Cell Line , 2022 .