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Mechanisms of humoral immune response against Pseudomonas aeruginosa biofilm infection in cystic fibrosis

  • Renan Marrichi Mauch
    Affiliations
    Department of Clinical Pathology, School of Medical Sciences, University of Campinas, Brazil
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  • Peter Østrup Jensen
    Affiliations
    Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, Denmark
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  • Claus Moser
    Affiliations
    Department of Clinical Microbiology, Rigshospitalet (Copenhagen University Hospital), Denmark
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  • Carlos Emilio Levy
    Affiliations
    Department of Clinical Pathology, School of Medical Sciences, University of Campinas, Brazil

    Laboratory of Microbiology, Division of Clinical Pathology, Hospital de Clínicas (Campinas University Hospital), Brazil
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  • Niels Høiby
    Correspondence
    Corresponding author at: Rigshospitalet (Session 9301), Juliane Maries Vej, 2100 Copenhagen, Capital Region, Denmark.
    Affiliations
    Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, Panum Institute, University of Copenhagen, Denmark

    Department of Clinical Microbiology, Rigshospitalet (Copenhagen University Hospital), Denmark
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Open ArchivePublished:October 21, 2017DOI:https://doi.org/10.1016/j.jcf.2017.08.012

      Abstract

      P. aeruginosa chronic lung infection is the major cause of morbidity and mortality in patients with cystic fibrosis (CF), and is characterized by a biofilm mode of growth, increased levels of specific IgG antibodies and immune complex formation. However, despite being designed to combat this infection, such elevated humoral response is not associated with clinical improvement, pointing to a lack of anti-pseudomonas effectiveness. The mode of action of specific antibodies, as well as their structural features, and even the background involving B-cell production, stimulation and differentiation into antibody-producing cells in the CF airways are poorly understood. Thus, the aim of this review is to discuss studies that have addressed the intrinsic features of the humoral immune response and provide new insights regarding its insufficiency in the CF context.

      Keywords

      1. Introduction

      Pulmonary disease is the preponderant manifestation in cystic fibrosis (CF), where a dehydration in the airway surface liquid (ASL) leads to mucus accumulation in the lower airways, impairing the mucociliary clearance (MCC) and facilitating colonization and infection of the lower airway environment by bacterial pathogens, especially Pseudomonas aeruginosa, the major morbidity and mortality cause in the CF population [
      • Stoltz D.
      • Meyerholz D.K.
      • Welsh M.J.
      Origins of cystic fibrosis lung disease.
      ]. P. aeruginosa infection starts with a non-mucoid planktonic variant, which is frequently succeeded by colonization-treatment-recolonization cycles, marked by intermittent bacterial isolations in microbiological respiratory tract cultures. This can further develop into a chronic stage of infection, marked by a mucoid biofilm-growing P. aeruginosa. Biofilm formation provides protection against the immune system (Fig. 1, Fig. 2) and antibiotics, as well as provoking an intense inoperative tissue-damaging inflammatory response, rendering P. aeruginosa practically impossible to eradicate in this phase of infection [
      • Folkesson A.
      • Jelsbak L.
      • Yang L.
      • Johansen H.K.
      • Ciofu O.
      • Høiby N.
      • et al.
      Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective.
      ,
      • Moradali M.F.
      • Ghods S.
      • Rehm B.H.A.
      Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence.
      ]. Moreover, the ionic unbalance caused by the CFTR defects leads to changing chemical characteristics of the ASL, where an acidified environment is formed, reducing or inactivating defensins, lysozymes and lactoferrins, natural defense molecules of the primary airway barrier, making bacterial killing even more difficult [
      • Abou Alaiwa M.H.
      • Reznikov L.R.
      • Gansemer N.D.
      • Sheets K.A.
      • Horswill A.R.
      • Stoltz D.A.
      • et al.
      pH modulates the activity and synergism of the airway surface liquid antimicrobials β-defensin-3 and LL-37.
      ,
      • Berkebile A.R.
      • McCray P.B.
      Effects of airway surface liquid pH on host defense in cystic fibrosis.
      ].
      Fig. 1.
      Fig. 1Mucus with a P. aeruginosa biofilm surrounded by polymorphonuclear neutrophils (PMN) and IgG antibodies specific to P. aeruginosa antigens, such as alginate, LPS and proteins. Immune complexes are formed between P. aeruginosa antigens and IgG, which activate complement and attract PMNs. Activated PMNs consume oxygen and liberate ROS, proteases and DNA, starting the inflammatory reaction. Such inflammatory reaction consumes all oxygen in mucus, which becomes anaerobic, impairing the PMNs' ability to phagocytose and kill P. aeruginosa in sputum due to the oxygen starvation needed for PMN activity, and leading to a frustrated phagocytosis. The pronounced IgG antibody response occurs especially due to the high NF-κB production by CF airway epithelial cells (AECs), and to the Th2-skewing of the adaptive immune reponse, resulting in hyperproduction of proinflammatory cytokines, like IL-5 and IL-8, and low production of anti-inflammatory cytokines, like IL-10 and INF-γ. Since the PMNs' ability is impaired, this hyperinflammatory response leads to tissue damage rather than killing P. aeruginosa biofilms. IgA is produced in the Bronchi-Associated Lymphoyd Tissue (BALT) submucosa (1), where it combines with the secretory component, yielding sIgA, which is exported through the epithelal cells to the airway surface liquid (ASL) (2), preventing P. aeruginosa to attach to the epithelial cells, in a macrophage-mediated non-phlogistic response, thereby not contributing to airway inflammation.
      Fig. 2.
      Fig. 2Fluorescence in situ hybridization staining of P. aeruginosa in a biofilm from an explanted chronically infected CF lung. Bacteria are stained red and the surrounding PMNs are stained blue (the nuclei). Extracted from Bjarnsholt et al.
      [
      • Bjarnsholt T.
      • Jensen P.Ø.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ]
      .
      Still, a number of studies in the last two decades have shown that not only the primary immune barriers are impaired in the CF airways, but also the cellular and signaling artifacts of the immune system as a whole is involved [
      • Yonker L.M.
      • Cigana C.
      • Hurley B.P.
      • Bragonzi A.
      Host-pathogen interplay in the respiratory environment of cystic fibrosis.
      ]. An interesting fact is that such impairment does not necessarily mean deficiency, but mostly overproduction of several immune components. CF airway epithelial cells (AECs), when challenged with P. aeruginosa, show increased activation of the Nuclear Transcription Factor – Kappa B (NF-κB), which leads to overproduction of pro-inflammatory cytokines interleukin (IL-) 6 and 8, and Tumor Necrosis Factor Alpha (TNF-α) disproportionately to the bacterial load in the airways, when compared with normal AECs. NF-κB overproduction also reduces the synthesis of glutathione, an antioxidant peptide that neutralizes reactive oxygen species (ROS) and increases the production of inflammatory mediators like Prostaglandin E2 and Cycloxygenase-2 (COX-2) [
      • Ziady A.G.
      • Hansen J.
      Redox balance in cystic fibrosis.
      ]. CF AECs also produce a decreased amount of interferon-gamma (IFN-γ), causing upregulation of IL-33, which, like IL-6 and IL-8, are neutrophil chemoattractants [
      • Parker D.
      • Cohen T.S.
      • Alhede M.
      • Harfenist B.S.
      • Martin F.J.
      • Prince A.
      Induction of type I interferon signaling by Pseudomonas aeruginosa is diminished in cystic fibrosis epithelial cells.
      ]. Consequently, neutrophils extravasate to the airways and are activated, releasing substantial levels of proteases and cationic peptides, deteriorating the airway surface and permeability [
      • Twigg M.S.
      • Brockbank S.
      • Lowry P.
      • FitzGerald S.P.
      • Taggart C.
      • Weldon S.
      The role of serine proteases and antiproteases in the cystic fibrosis lung.
      ]. Under normal conditions, neutrophils undergo apoptosis and are swept from the airways by cough clearance or phagocytosis by alveolar macrophages (AM). In the CF context, notwithstanding the impaired MCC, macrophages' ability to phagocyte apoptotic neutrophils is also decreased; thus, neutrophils undergo necrosis rather than apoptosis, releasing their intracellular content in the airways, including chemoattractants (attracting other neutrophils to the inflammation site, thus perpetuating the immune response), DNA content (that serves as a biofilm matrix), proteases and ROS (damaging the airway structure) [
      • McCaslin C.A.
      • Petrusca D.N.
      • Poirier C.
      • Serban K.A.
      • Anderson G.G.
      • Petrache I.
      Impact of alginate-producing Pseudomonas aeruginosa on alveolar macrophage apoptotic cell clearance.
      ,
      • Bruscia E.M.
      • Bonfield T.L.
      Cystic fibrosis lung immunity: the role of the macrophage.
      ].
      In the adaptive immune response, more specifically in the T-cell response, a predilection to mount a Th2-skewed response is observed, with upregulation of IL-5 and IL-13, and higher B-cell sensitivity to IL-4. Such response is well known to fight parasites, not being efficient against bacterial pathogens like P. aeruginosa [
      • Yonker L.M.
      • Cigana C.
      • Hurley B.P.
      • Bragonzi A.
      Host-pathogen interplay in the respiratory environment of cystic fibrosis.
      ]. There are evidences that CF Th cells also produce low levels of IFN-γ (a Th1-associated cytokine) and elevated levels of IL-10, a Th2-associated cytokine with ability to downregulate IFN-γ and to decrease co-stimulatory molecules in macrophages, thereby hindering antigen presentation and preventing a proper immune response to P. aeruginosa, Aspergillus fumigatus and other CF pathogens [
      • Moss R.B.
      • Hsu Y.P.
      • Olds L.
      Cytokine dysregulation in activated cystic fibrosis (CF) peripheral lymphocytes.
      ,
      • Casaulta C.
      • Schöni M.H.
      • Weichel M.
      • Crameri R.
      • Jutel M.
      • Daigle I.
      • et al.
      IL-10 controls aspergillus fumigatus- and Pseudomonas aeruginosa-specific T-cell response in cystic fibrosis.
      ,
      • Hartl D.
      • Griese M.
      • Kappler M.
      • Zissel G.
      • Reinhardt D.
      • Rebhan C.
      • et al.
      Pulmonary TH2 response in Pseudomonas aeruginosa-infected patients with cystic fibrosis.
      ]. It has been suggested that the Th1 response can be protective against P. aeruginosa infection and confer resistance to reinfection [
      • Moser C.
      • Jensen P.O.
      • Kobayashi O.
      • Hougen H.P.
      • Song Z.
      • Rygaard J.
      • et al.
      Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response.
      ]. IFN-γ treatment in a chronic lung infection rat model using alginate embedded P. aeruginosa changed the pulmonary response from an inflammation dominated by neutrophils to mononuclear leucocytes [
      • Johansen H.K.
      • Hougen H.P.
      • Rygaard J.
      • Høiby N.
      Interferon-gamma (IFN-gamma) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats.
      ]. Moreover, IFN-γ induction of alveolar macrophages may mediate removal of apoptotic neutrophils, preventing further increased inflammation due to progression into necrosis [
      • Moser C.
      • Pedersen H.T.
      • Lerche C.J.
      • Kolpen M.
      • Line L.
      • Thomsen K.
      • et al.
      Biofilms and host response: helpful or harmful?.
      ].
      The Th17 response has also been recently addressed in CF as having the ability to stimulate the neutrophil mobiliser G-CSF and the neutrophil chemoattractant IL-8, thus contributing to the pulmonary pathology during chronic P. aeruginosa lung infection. The rate of naive T-cells that differentiate into Th17 cells is almost twice as high as in healthy subjects. This amount correlates with the severity of the lung disease, especially caused by P. aeruginosa, suggesting that IL-17 and associated cytokines can serve both as biomarkers for early detection of the disease and potential therapeutic targets [
      • Tiringer K.
      • Treis A.
      • Fucik P.
      • Gona M.
      • Gruber S.
      • Renner S.
      • et al.
      A Th17- and Th2-skewed cytokine profile in cystic fibrosis lungs represents a potential risk factor for Pseudomonas aeruginosa infection.
      ,
      • Kushwah R.
      • Gagnon S.
      • Sweezey N.B.
      Intrinsic predisposition of naïve cystic fibrosis T cells to differentiate towards a Th17 phenotype.
      ,
      • Bayes H.K.
      • Bicknell S.
      • MacGregor G.
      • Evans T.J.
      T helper cell subsets specific for Pseudomonas aeruginosa in healthy individuals and patients with cystic fibrosis.
      ]. Furthermore, a Th2-Th17 axis in CF may predispose for development P. aeruginosa lung infection [
      • Moser C.
      • Pedersen H.T.
      • Lerche C.J.
      • Kolpen M.
      • Line L.
      • Thomsen K.
      • et al.
      Biofilms and host response: helpful or harmful?.
      ,
      • Tiringer K.
      • Treis A.
      • Fucik P.
      • Gona M.
      • Gruber S.
      • Renner S.
      • et al.
      A Th17- and Th2-skewed cytokine profile in cystic fibrosis lungs represents a potential risk factor for Pseudomonas aeruginosa infection.
      ,
      • Bayes H.K.
      • Bicknell S.
      • MacGregor G.
      • Evans T.J.
      T helper cell subsets specific for Pseudomonas aeruginosa in healthy individuals and patients with cystic fibrosis.
      ,
      • Decraene A.
      • Willems-Widyastuti A.
      • Kasran A.
      • De Boeck K.
      • Bullens D.M.
      • Dupont L.J.
      Elevated expression of both mRNA and protein levels of IL-17A in sputum of stable cystic fibrosis patients.
      ].

      2. B-cells, antibodies and the humoral immune response in CF

      Although the elucidation of CF immunological disorders has been facilitating the understanding of the lung disease natural history, the efficacy of the humoral immune response remains a poorly explored field concerning the pathogen-immune system interaction. In P. aeruginosa infection, particularly, the high antibody level followed by immune complex (IC) formation is a hallmark (Fig. 1). Pioneer studies had already shown that the level of specific P. aeruginosa precipitating antibodies were increased in the presence of the mucoid phenotype and associated with poor prognosis [
      • Høiby N.
      • Flensborg E.W.
      • Beck B.
      • Friis B.
      • Jacobsen S.V.
      • Jacobsen L.
      Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis.
      ]. From then on, the antibody response with diagnostic purposes was tested against a broad variety of P. aeruginosa antigens, showing frequently enhanced antibody levels (mainly IgG), even in patients without P. aeruginosa chronic lung infection [
      • Mauch R.M.
      • Rossi C.L.
      • Ribeiro J.D.
      • Ribeiro A.F.
      • Nolasco da Silva M.T.
      • Levy C.E.
      Assessment of IgG antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis by an enzyme-linked immunosorbent assay (ELISA).
      ]. The response against particular antigens appears to depend on the infection stage, with some antigens provoking a more intense response in the acute phase, while others are more targeted during the chronic phase [
      • Mauch R.M.
      • Levy C.E.
      Serum antibodies to Pseudomonas aeruginosa in cystic fibrosis as a diagnostic tool: a systematic review.
      ]. However, the relation between response intensity and pulmonary damage, as well as the lack of the association between humoral response and clinical improvement are well reported [
      • Brett M.M.
      • Ghoneim A.T.
      • Littlewood J.M.
      Prediction and diagnosis of early Pseudomonas aeruginosa infection in cystic fibrosis: a follow-up study [published erratum appears in J Clin Microbiol 1989 Jan 27 (1):230].
      ,
      • West S.E.H.
      • Zeng L.
      • Lee B.L.
      • Kosorok M.R.
      • Laxova A.
      • Rock M.J.
      • et al.
      Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors.
      ]. Besides, antibiotic treatment may decrease anti-Pseudomonas antibody production, but even after treatment, there is an increased risk of recurrent infection if patients display high antibody levels [
      • Anstead M.
      • Heltshe S.L.
      • Khan U.
      • Barbieri J.T.
      • Langkamp M.
      • Döring G.
      • et al.
      Pseudomonas aeruginosa serology and risk for re-isolation in the EPIC trial.
      ].
      Despite the evidences of the inefficiency of humoral response to mediate the clearance of infection from the airways, the mode of action of specific IgG anti-Pseudomonas antibodies, as well as their subclass distribution and the scenario involving B-cell production, stimulation and differentiation into plasma cells in the airways remains poorly studied in CF.

      2.1 B-cells in CF

      B-cells primarily rise from hematopoietic stem cells and their development is marked by a continuum of stages that starts in the primary lymphoid tissues – mainly the bone marrow (BM). B-cell maturation progresses in sequential steps in the BM and, after this, the B-cells migrate to and complete the maturation in the secondary lymphoid tissues (SLT) - e.g. lymph node and spleen [
      • Lebien T.W.
      • Tedder T.F.
      B lymphocytes : how they develop and function.
      ]. Antigen-induced activation and differentiation in the SLTs are mediated by dynamic changes in gene expression that originate in the germinal center (GC) reaction. GCs are transient structures formed inside the SLT and dedicated to the production of high affinity antibodies, where the B-cells expressing such membrane bound antibodies mature, undergo stimulation and develop into antibody-producing cells (plasma cells) or memory B-cells. Developing B-cells progresses through rearrangement of gene segments of the light and heavy immunoglobulin (Ig) chains (V, D and J) of pre- and pro-B-cells, thus culminating in the expression of the mature B-cell receptor (BCR) (IgD or IgM) in the cell surface, which can bind antigens (Fig. 3). The maturation steps depend on close interactions between developing B-cells and BM stromal cells, which provide critical adhesive integrins, growth factors, chemokines and cytokines. Immature B-cells that leave the BM towards the SLTs have the maturation process guided by BCR signals, B-cell Activating Factor (BAFF), and expression of transcription factors, like NOTCH2 and BTK [
      • Hoffman W.
      • Lakkis F.G.
      • Chalasani G.
      B cells, antibodies, and more.
      ].
      Fig. 3.
      Fig. 3After synthesis in the bone marrow, naïve B cells are activated in the secondary lymphoid organs (e.g. CF BALT) during antigen presentation by an antigen-presenting cell (e.g. dendritic cell), and interaction with the antigen. B cells then proliferate and generate plasma cells, which produce antibodies (e.g. IgG and IgA). Antigen-specific naïve B cells can differentiate within secondary lymphoid tissue into short-lived low-affinity plasma cells or undergo a rapid proliferative phase known as the germinal center (GC) reaction, where clonal expansion and affinity/avidity maturation take place, yielding either memory B cells or high-affinity long-lived plasma cells. When memory B cells are re-activated by a specific antigen, they quickly respond to the cognate antigen, proliferate and differentiate into the high-affinity long-lived antibody producing plasma cells. CF antibodies undergo deficient affinity/avidity maturation
      [
      • Mauch R.M.
      • Nørregard L.L.
      • Jensen P.Ø.
      • Levy C.E.
      • Høiby N.
      76 avidity of IgG antibodies and opsonic quality of serum samples against Pseudomonas aeruginosa during chronic lung infection in cystic fibrosis.
      ]
      . This may be due to the continous pronounced antigen stimulation, which takes place over decades in well-treated patients, where both high-affinity and low affinity clones are activated. This is completely different from the controlled situation during vaccination, where small amounts of antigens are repeatedly administered to obtain protective immunity and where only high affinity clones are activated. IgA is mainly transported to the ASL to keep bacteria and their products at a distance from the epithelial surface in order to prevent damage to the epithelial surface (see ). IgG, on the other hand, is then transported to the interstial fluid and to the blood, reaching the respiratory zone (alveoli) (1), where it binds the bacteria present in this zone, contributing to the inflammatory response, attracting macrophages and neutrophils, leading to secretion of proinflammatory cytokines. These cytokines attract more neutrophils through transmigration from the capillary endothelia to the alveolar space (2). Neutrophils then produce proteases and ROS, provoking a gradual and extensive lung injury.
      High numbers of B-cells have been documented in CF patients infected with P. aeruginosa, and low levels in patients under treatment for pulmonary exacerbation. Patients with persistent infection with P. aeruginosa showed high levels of immunoglobulins in ASL, mainly IgA; however, this response appears to be insufficient to remove the bacteria, prevent chronic infection or acquisition of new strains [
      • Yonker L.M.
      • Cigana C.
      • Hurley B.P.
      • Bragonzi A.
      Host-pathogen interplay in the respiratory environment of cystic fibrosis.
      ]. In another report, CF patients showed a significantly higher spontaneous formation of plaque-forming cells (which reflect the B-cell differentiation into plasma cells in vivo), when compared with normal individuals after challenge with mitogen or staphyloccoci, as well as a significant impairment in the B-cell differentiation in response to the polyclonal activation in vitro, which could not be explained by the increased numbers of adherent suppressor cells or by the presence of suppression enhanced by T-cells [
      • Sorensen R.U.
      • Ruuskanen O.
      • Miller K.
      • Stern R.C.
      B-lymphocyte function in cystic fibrosis.
      ]. Neill et al. [
      • Neill D.R.
      • Saint G.L.
      • Bricio-Moreno L.
      • Fothergill J.L.
      • Southern K.W.
      • Winstanley C.
      • et al.
      The B lymphocyte differentiation factor (BAFF) is expressed in the airways of children with CF and in lungs of mice infected with Pseudomonas aeruginosa.
      ] recently showed, for the first time, the kinetic of B-cell responses to chemoattractant factors and the kinetic of B-cell differentiation, both following P. aeruginosa infection and in relation to the B-cell recruitment, in a CF murine model. P. aeruginosa infection showed association with elevated levels of BAFF and another B-cell chemoattractants, like CXCL13, CCL19 and CCL21. An elevated BAFF level was also found in bronchoalveolar lavage (BAL) of CF pediatric patients, irrespective of absence or presence of P. aeruginosa infection, implying that the expression is not specific for this pathogen and can be a feature of the CF airways, and suggesting that not only the B-cells recruited to the airway, but the local environment as well, is able to support the B-cell survival, differentiation and antibody production; however, persistence of P. aeruginosa infection suggests that this response is ineffective. Still, there are no studies confirming whether there is any failure in B-cell production, regulation and proliferation in CF.

      2.2 Functionality of antibody response in CF

      2.2.1 Immune complex formation

      IC formation during P. aeruginosa lung infection in sputum (Fig. 1) and serum from CF patients is long-established [
      • McFarlane H.
      • Holzel A.
      • Brenchley P.
      • Allan J.D.
      • Wallwork J.C.
      • Singer B.E.
      • et al.
      Immune complexes in cystic fibrosis.
      ,
      • Schiøtz P.O.
      • Nietsen H.
      • Høiby N.
      • Glikmann G.
      • Svehag S.E.
      Immune complexes in the sputum of patients with cystic fibrosis suffering from chronic Pseudomonas aeruginosa lung infection.
      ]. A significantly higher IgG2 concentration was reported in sera of CF patients chronically infected with P. aeruginosa, as well as higher mean levels of circulating immune complexes (CIC). CIC removal was accompanied by significant decrease in IgG2 concentration without changes in other subclasses, and by a significant increase of both macrophage and neutrophil-mediated phagocytosis, therefore suggesting IgG2 to significantly contribute to CIC formation and to have antiphagocytic activity through direct inhibition in CF [
      • Hornick D.B.
      • Fick Jr., R.B.
      The immunoglobulin G subclass composition of immune complexes in cystic fibrosis.
      ]. ICs in sputum of CF patients chronically infected with P. aeruginosa were found to be basically composed of lipopolysaccharide (LPS), IgG1–4, IgA and IgM. Still, high CIC concentration was shown to be positively correlated with the TNF-α amount. In vitro analyses showed that the ICs containing LPS stimulated a higher TNF-α release than LPS alone, and also induced oxidative stress in neutrophils [
      • Kronborg G.
      • Fomsgaard A.
      • Galanos C.
      • Freudenberg M.
      • Høiby N.
      Antibody responses to lipid A, core, and O sugars of the Pseudomonas aeruginosa lipopolysaccharide in chronically infected cystic fibrosis patients.
      ]. One study found that C4 activation was highly associated with CIC [
      • Moss R.B.
      • Hsu Y.P.
      • Lewiston N.J.
      • Curd J.G.
      • Milgrom H.
      • Hart S.
      • et al.
      Association of systemic immune complexes, complement activation, and antibodies to Pseudomonas aeruginosa lipopolysaccharide and exotoxin A with mortality in cystic fibrosis.
      ], but two other did not find any relation between CIC and complement activation [
      • Moss R.B.
      • Lewiston N.J.
      Immune complexes and humoral response to Pseudomonas aeruginosa in cystic fibrosis.
      ,
      • Church J.A.
      • Jordan S.C.
      • Keens T.G.
      • Wang C.I.
      Circulating immune complexes in patients with cystic fibrosis.
      ].
      Conflicting results were obtained in attempts to correlate IC formation and level with clinical state. CF patients with acute lung disease had higher CIC formation than stable patients [
      • Church J.A.
      • Jordan S.C.
      • Keens T.G.
      • Wang C.I.
      Circulating immune complexes in patients with cystic fibrosis.
      ] and higher CIC levels were found in patients with worst clinical states in three studies, being associated with worst clinical and X-ray scores, lower lung function results, lower weight, and higher anti-P. aeruginosa IgG levels [
      • Hodson M.E.
      • Beldon I.
      • Batten J.C.
      Circulating immune complexes in patients with cystic fibrosis.
      ,
      • Van Bever H.P.
      • Gigase P.L.
      • De Clerck L.S.
      • Bridts C.H.
      • Franckx H.
      • Stevens W.J.
      Immune complexes and Pseudomonas aeruginosa antibodies in cystic fibrosis.
      ,
      • Lagacé J.
      • Mercier J.
      • Frechette M.
      • Fournier D.
      • Dubreuil M.
      • Lamarre A.
      • et al.
      Circulating immune complexes, antibodies to Pseudomonas aeruginosa, and pulmonary status in cystic fibrosis.
      ]. A multifactorial analysis with 10 years of follow-up [
      • Disis M.L.
      • McDonald T.L.
      • Colombo J.L.
      • Kobayashi R.H.
      • Angle C.R.
      • Murray S.
      Circulating immune complexes in cystic fibrosis and their correlation to clinical parameters.
      ] reported correlation between CIC and only four of 38 clinical parameters – low National Institute of Health (NIH) score, higher age, low rate of respiratory peak flow and high total IgG level. There was no correlation between CIC and severity of individual pulmonary exacerbation; however, all patients who died during the follow-up had higher CIC levels, comparing with those who survived. Two studies observed association between CIC evidence and/or formation with mortality [
      • Moss R.B.
      • Hsu Y.P.
      • Lewiston N.J.
      • Curd J.G.
      • Milgrom H.
      • Hart S.
      • et al.
      Association of systemic immune complexes, complement activation, and antibodies to Pseudomonas aeruginosa lipopolysaccharide and exotoxin A with mortality in cystic fibrosis.
      ,
      • Wisnieski J.J.
      • Todd E.W.
      • Fuller R.K.
      • Jones P.K.
      • Dearborn D.G.
      • Boat T.F.
      • et al.
      Immune complexes and complement abnormalities in patients with cystic fibrosis. Increased mortality associated with circulating immune complexes and decreased function of the alternative complement pathway.
      ], while other studies did not find association between ICs and colonization or response to P. aeruginosa, Staphylococcus aureus, Haemophilus influenzae and streptococcal species, nor correlation of ICs with severity or progression of lung disease, pulmonary exacerbations, or non-specified humoral and cellular functions [
      • Moss R.B.
      • Lewiston N.J.
      Immune complexes and humoral response to Pseudomonas aeruginosa in cystic fibrosis.
      ,
      • Berdischewsky M.
      • Pollack M.
      • Young L.S.
      • Chia D.
      • Osher A.B.
      • Barnett E.V.
      Circulating immune complexes in cystic.
      ,
      • Pitcher-Wilmott R.W.
      • Levinsky R.J.
      • Matthew D.J.
      Circulating soluble immune complexes containing pseudomonas antigens in cystic fibrosis.
      ]. Proteolytic cleavage of ICs was suggested to contribute to the failure to detect ICs in CF and therefore to the lack of correlation with disease severity in some studies [
      • Döring G.
      • Albus A.
      • Høiby N.
      Immunologic aspects of cystic fibrosis.
      ].

      2.2.2 Opsonization capacity and phagocytic killing

      Derived from the greek word opson (condiment), opsonization is the process by which a foreign particle, particularly a microbe, is coated with plasma proteins (opsonins) aiming to facilitate the microbe attachment and internalization by a professional phagocytic cell. Overall, the process refers to the microbe coating with Ig molecules (antibodies) that are specific for the antigenic determinants of that organism, or with complement proteins (particularly C3b) that are deposited upon the surface of the microorganism both through the classic or alternative activation pathway. The presence of these proteins in the microbial surface facilitates its sequential interaction with the Ig receptors (Fc receptors) or complement receptors in the phagocyte surface. These interactions result in encirclement of the particle by the cytoplasmic membrane of the phagocytic cell, until the particle is contained in a membrane-bound vacuole (phagosome) within the cell [
      • Czuprynski C.J.
      Opsonization and phagocytosis.
      ].
      Early reports found inability of bactericidal activity in sera from CF patients with advanced lung disease, particularly against autologous P. aeruginosa isolates, suggesting the presence of “bactericidal block” antibodies [
      • Høiby N.
      • Schiøtz P.O.
      Immune complex mediated tissue damage in the lungs of cystic fibrosis patients with chronic Pseudomonas aeruginosa infection.
      ,
      • Thomassen M.J.
      • Demko C.A.
      Serum bactericidal effect on Pseudomonas aeruginosa isolates from cystic fibrosis patients.
      ], and attributing this defect to IgG [
      • Guttman R.M.
      • Waisbren B.A.
      Bacterial blocking activity of specific IgG in chronic Pseudomonas aeruginosa infection.
      ]. Such findings headed the investigation of the opsonic capacity of CF sera, revealing a deficiency of sera from patients infected with P. aeruginosa to promote phagocytosis (in relation to normal individuals and patients without P. aeruginosa infection) particularly by alveolar macrophages [
      • LeBlanc C.M.A.
      • Bortolussi R.
      • Issekutz A.C.
      • Gillespie T.
      Opsonization of mucoid and non-mucoid Pseudomonas aeruginosa by serum from patients with cystic fibrosis assessed by a chemiluminescence assay.
      ,
      • Moss R.B.
      • Hsu Y.P.
      • Sullivan M.M.
      • Lewiston N.J.
      Altered antibody isotype in cystic fibrosis: possible role in opsonic deficiency.
      ,
      • Thomassen M.J.
      • Boxerbaum B.
      • Demko C.A.
      • Kuchenbrod P.J.
      • Dearborn D.G.
      • Wood R.E.
      Inhibitory effect of cystic fibrosis serum on Pseudomonas phagocytosis by rabbit and human alveolar macrophages.
      ,
      • Thomassen M.J.
      • Demko C.A.
      • Wood R.E.
      • Tandler B.
      • Dearborn D.G.
      • Boxerbaum B.
      • et al.
      Ultrastructure and function of alveolar macrophages from cystic fibrosis patients.
      ], but not by PMNs [
      • LeBlanc C.M.A.
      • Bortolussi R.
      • Issekutz A.C.
      • Gillespie T.
      Opsonization of mucoid and non-mucoid Pseudomonas aeruginosa by serum from patients with cystic fibrosis assessed by a chemiluminescence assay.
      ,
      • Pier G.B.
      • Saunders M.J.
      • Ames P.
      • Edwards M.S.
      • Auerbach H.
      • Goldfarb J.
      • et al.
      Opsonophagocytic killing antibody to Pseudomonas aeruginosa mucoid exopolysaccharide in older noncolonized patients with cystic fibrosis.
      ]. Complement activation is reported as being normal [
      • Thomassen M.J.
      • Demko C.A.
      Serum bactericidal effect on Pseudomonas aeruginosa isolates from cystic fibrosis patients.
      ,
      • LeBlanc C.M.A.
      • Bortolussi R.
      • Issekutz A.C.
      • Gillespie T.
      Opsonization of mucoid and non-mucoid Pseudomonas aeruginosa by serum from patients with cystic fibrosis assessed by a chemiluminescence assay.
      ,
      • Moss R.B.
      • Hsu Y.P.
      • Sullivan M.M.
      • Lewiston N.J.
      Altered antibody isotype in cystic fibrosis: possible role in opsonic deficiency.
      ,
      • Pier G.B.
      • Saunders M.J.
      • Ames P.
      • Edwards M.S.
      • Auerbach H.
      • Goldfarb J.
      • et al.
      Opsonophagocytic killing antibody to Pseudomonas aeruginosa mucoid exopolysaccharide in older noncolonized patients with cystic fibrosis.
      ,
      • Moss R.B.
      The role of IgG subclass antibodies in chronic infection: the case of cystic fibrosis.
      ,
      • Pressler T.
      • Jensen E.T.
      • Espersen F.
      • Pedersen S.S.
      • Høiby N.
      High levels of complement-activation capacity in sera from patients with cystic fibrosis correlate with high levels of IgG3 antibodies to Pseudomonas aeruginosa antigens and poor lung function.
      ] and opsonic capacity appeared to include a non-complement cofactor for optimal activity [
      • Moss R.B.
      • Hsu Y.P.
      • Sullivan M.M.
      • Lewiston N.J.
      Altered antibody isotype in cystic fibrosis: possible role in opsonic deficiency.
      ].
      Immunoglobulin activity, on the other hand, was suggested to be deficient and even inhibitory, confirming previous reports. A series of studies by Moss et al. [
      • Moss R.B.
      • Hsu Y.P.
      • Lewiston N.J.
      • Curd J.G.
      • Milgrom H.
      • Hart S.
      • et al.
      Association of systemic immune complexes, complement activation, and antibodies to Pseudomonas aeruginosa lipopolysaccharide and exotoxin A with mortality in cystic fibrosis.
      ,
      • Moss R.B.
      • Hsu Y.P.
      • Sullivan M.M.
      • Lewiston N.J.
      Altered antibody isotype in cystic fibrosis: possible role in opsonic deficiency.
      ,
      • Moss R.B.
      The role of IgG subclass antibodies in chronic infection: the case of cystic fibrosis.
      ,
      • Eichler I.
      • Joris L.
      • Hsu Y.P.
      • Van Wye J.
      • Bram R.
      • Moss R.
      Nonopsonic antibodies in cystic fibrosis. Pseudomonas aeruginosa lipopolysaccharide-specific immunoglobulin G antibodies from infected patient sera inhibit neutrophil oxidative responses.
      ] found that CF patients with P. aeruginosa infection displayed a broadly distributed hyperimmunoglobulinemia G, and even not infected patients were subject to have elevated IgG3 and IgG4 levels. The major antigenic determinant of the elevated IgG serum response was the P. aeruginosa LPS. Such elevation showed distribution among all IgG subclasses, with a shift towards IgG3. Notwithstanding, when serum opsonic capacity was compared with level of autologous antibodies, increased levels of IgG4, but not IgG1–3 were found in patients with poor serum opsonic activity. Moreover, an inverse correlation was found between IgG4 levels and opsonic activity, suggesting a blocking capacity of AM-mediated P. aeruginosa phagocytosis by antibodies of this particular isotype. In addition, patients who were not infected by P. aeruginosa had decreased IgG2 levels and increased IgG4 levels to LPS when compared with normal controls. IgG4 indeed has a poor opsonizing capacity and an efficient response of this subclass is not expected, since IgG2 is the subclass that better responds to polysaccharide antigens like LPS [
      • Moss R.B.
      The role of IgG subclass antibodies in chronic infection: the case of cystic fibrosis.
      ]. The low IgG2 levels raises the possibility of restricted immunodeficiency to polysaccharide antigens, with an attempt of compensatory shift towards IgG3 and IgG4 doomed to failure. Indeed, almost one decade later, it was documented that patients with high IgG3 levels to LPS, alginate and a mixed P. aeruginosa antigen (sonicate) are able to induce a high complement activation level that, however, is associated with a more aggressive lung damage, probably secondary to local IC formation and reflecting the inefficiency of this response [
      • Pressler T.
      • Jensen E.T.
      • Espersen F.
      • Pedersen S.S.
      • Høiby N.
      High levels of complement-activation capacity in sera from patients with cystic fibrosis correlate with high levels of IgG3 antibodies to Pseudomonas aeruginosa antigens and poor lung function.
      ].
      Although PMN-mediated phagocytosis induction has been reported as being normal, after P. aeruginosa opsonization with CF sera, chemiluminescence analyses found that, while opsonization of P. aeruginosa with P. aeruginosa-infected sera resulted in increased levels of PMN chemiluminescence, when corrected for equimolar P. aeruginosa LPS-specific IgG content, P. aeruginosa-infected sera displayed markedly lower opsonizing ability compared to P. aeruginosa-uninfected sera, normal sera, or normal serum-derived P. aeruginosa hyperimmune globulin [
      • Eichler I.
      • Joris L.
      • Hsu Y.P.
      • Van Wye J.
      • Bram R.
      • Moss R.
      Nonopsonic antibodies in cystic fibrosis. Pseudomonas aeruginosa lipopolysaccharide-specific immunoglobulin G antibodies from infected patient sera inhibit neutrophil oxidative responses.
      ]. Recent reports [
      • Thomsen K.
      • Christophersen L.
      • Bjarnsholt T.
      • Jensen P.
      • Moser C.
      • Høiby N.
      Anti-Pseudomonas aeruginosa IgY antibodies promote specific bacterial aggregation and internalization in polymorphonuclear neutrophils.
      ,
      • Thomsen K.
      • Christophersen L.
      • Jensen P.Ø.
      • Bjarnsholt T.
      • Moser C.
      • Høiby N.
      Anti-Pseudomonas aeruginosa IgY antibodies promote bacterial opsonization and augment the phagocytic activity of polymorphonuclear neutrophils.
      ] showed that P. aeruginosa opsonized with egg yolk antibodies (IgY), the analogue avian antibody for human IgG, enhanced the PMN-mediated respiratory burst and subsequent bacterial killing in vitro. Such effects were observed against different P. aeruginosa strains, and possibly reflect a decreased opsonizing capacity of CF IgG to head PMN-mediated phagocytosis.
      P. aeruginosa biofilm aggregates in the lungs and sputum of CF patients were shown to be surrounded by PMNs, but have never been observed inside PMNs (Fig. 1, Fig. 2). In contrary, planktonic P. aeruginosa cells were frequently seen inside the PMNs, engulfed, but presumably not destroyed. Since CF sputum becomes anaerobic, due to the PMN activity, these cells are deprived form oxygen and may not be able to produce a metabolic burst to kill the phagocytosed bacteria (Fig. 1) [
      • Bjarnsholt T.
      • Jensen P.Ø.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ] and thus functionally may be somewhat like PMNs from chronic granulomatous disease patients [
      • Holland S.M.
      Chronic granulomatous disease.
      ], who are able to phagocytose bacteria but not to kill catalase positive bacteria like e.g. S. aureus since they are defective in NADP-oxidase and, therefore, cannot produce O2. This is therefore a possible additional explanation for the functional inability of the immune system of CF patients to clear lung infections. As a matter of fact, our group has previously shown that hyperbaric oxygen treatment of O2-depleted P. aeruginosa biofilms could significantly enhance the bactericidal activity of ciprofloxacin on the bacteria, due to the increase of the diffusive supply for aerobic respiration during ciprofloxacin treatment of these biofilms, thereby showing the potential of combined treatments with hyperbaric oxygen [
      • Kolpen M.
      • Mousavi N.
      • Sams T.
      • Bjarnsholt T.
      • Ciofu O.
      • Moser C.
      • et al.
      Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment.
      ].

      2.2.3 Defective interaction among pathogen, antibodies and phagocytes

      Evidences of defective antibody functional capacity reported to date suggest that CF antibodies may have a low avidity (overall strength of an antigen-antibody complex) or affinity (strength of the interaction between the antigen binding site in the antibody and the antigen epitope) (Fig. 3) [
      • Gutiérrez J.
      • Maroto C.
      Are IgG antibody avidity assays useful in the diagnosis of infectious diseases? A review.
      ,
      • Dimitrov J.D.
      • Planchais C.
      • Roumenina L.T.
      • Vassilev T.L.
      • Kaveri S.V.
      • Lacroix-Desmazes S.
      Antibody polyreactivity in health and disease: statu variabilis.
      ].
      P. aeruginosa phagocytosis, by means of its AM-mediated internalization and opsonic killing, was shown to be reduced when the bacteria were opsonized with CF sera with high specific IgG levels, even when compared with non-opsonized bacteria. Besides, radioimmunoassay studies observing the AM-bacteria interaction found only a few bacteria attached to the AM membrane, thereby suggesting that a defect in the Fcγ portion of the Pseudomonas-specific IgG in CF interferes with the phagocytic function by preventing a proper receptor attachment and subsequent triggering of internalization. Such defect, with consequent decrease in the bacterial clearance, can lead to a prolonged circulation of ICs, causing tissue deposition and damage [
      • Fick R.B.
      • Naegel G.P.
      • Matthay R.A.
      • Reynolds H.Y.
      Cystic fibrosis Pseudomonas opsonins. Inhibitory nature in an in vitro phagocytic assay.
      ].
      A study addressing the immunization of CF patients and healthy adults with a LPS-based vaccine demonstrated that this vaccine elicited a significant increase in the total anti-LPS IgG levels and in the affinity of these antibodies. On the other hand, CF patients chronically colonized by P. aeruginosa (not immunized) showed affinity values at least 100 times lower than antibodies induced by the vaccine, despite of the elevated levels of naturally acquired anti-LPS IgG. These high affinity antibodies seemed to mediate the opsonophagocytic killing of P. aeruginosa by macrophages [
      • Bruderer U.
      • Cryz S.J.
      • Schaad U.B.
      • Deusinger M.
      • Que J.U.
      • Lang A.B.
      Affinity constants of naturally acquired and vaccine-induced anti-Pseudomonas aeruginosa antibodies in healthy adults and cystic fibrosis patients.
      ]. Other analysis [
      • Torensma R.
      • Fluit A.C.
      • Verhoef J.
      Reactivity of monoclonal antibodies to Pseudomonas aeruginosa isolates from hospitalized adults and patients with cystic fibrosis.
      ] found that P. aeruginosa isolates recovered from CF patients were reactive mainly with a monoclonal antibody directed to the outer core of the P. aeruginosa LPS, suggesting reactivity to LPS as having potential value in P. aeruginosa eradication in these patients. Similar conclusions were proposed by Lang et al. [
      • Lang A.B.
      • Schaad U.B.
      • Rüdeberg A.
      • Wedgwood J.
      • Que J.U.
      • Fürer E.
      • et al.
      Effect of high-affinity anti-Pseudomonas aeruginosa lipopolysaccharide antibodies induced by immunization on the rate of Pseudomonas aeruginosa infection in patients with cystic fibrosis.
      ], who found association between P. aeruginosa infection and absence of high affinity anti-LPS antibodies. The frequency of infection was significantly higher among patients who were not vaccinated with LPS and vaccinated patients who failed to mount a high affinity response, than among immunized patients. Most P. aeruginosa-positive respiratory cultures were obtained from specimens from immunized patients only after the loss of the high affinity circulating anti-LPS antibodies, which occurred between the second and third years (out of four years) of observation. No such correlation was found when total anti-P. aeruginosa LPS antibody levels versus infection rate were analyzed. Mucoid P. aeruginosa was cultured from a single vaccinated patient (out of four patients) who failed to mount a high affinity response, while six (out of 10) non-vaccinated patients become infected with the mucoid variants. Therefore, it was suggested that the presence of high affinity anti-LPS antibodies can act to keep the bacterial load below a critical level needed for the phenotypic switch.
      IgG avidity against P. aeruginosa exotoxin A (ExoA) was shown to be decreased in CF patients with P. aeruginosa chronic lung infection, despite being significantly different from patients without infection and healthy subjects. Also, no increase in anti-ExoA IgG avidity was seen in 6/8 chronically infected patients after a period of six months of follow-up [
      • Polanec J.
      • Patzer J.
      • Grzybowski J.
      • Strukelj M.
      • Pavelic Z.P.
      Amount and avidity of IgG antibodies to Pseudomonas aeruginosa exotoxin a antigen in cystic fibrosis patients.
      ]. A possible explanation is the long term exposure to the antigen via CF lungs, which can cause a specific suppression of the IgG response [
      • Brauner A.
      • Cryz S.J.
      • Granström M.
      • Hanson H.S.
      • Löfstrand L.
      • Strandvik B.
      • et al.
      Immunoglobulin G antibodies to Pseudomonas aeruginosa lipopolysaccharides and exotoxin A in patients with cystic fibrosis or bacteremia.
      ]. In a cohort of 11 CF patients with poor lung function and 9 patients with good lung function, all patients developed increasing levels of antibodies against P. aeruginosa beta-lactamase and against the 60–65 kDa heat shock protein (anti-GroEL), but no avidity maturation of these antibodies was observed. In patients with good lung function, the avidity of anti-beta-lactamase antibodies was higher than in patients with poor lung function, suggesting a more efficient inhibition of the beta-lactamase by these antibodies, resulting in better lung function [
      • Ciofu O.
      • Petersen T.D.
      • Jensen P.Ø.
      • Høiby N.
      Avidity of anti-P. aeruginosa antibodies during chronic infection in patients with cystic fibrosis.
      ].
      In recurrent Staphylococcus aureus infections, high levels of antibodies to a variety of antigens are seen in CF, including against the polysaccharide capsule, suggesting the presence of virulence factors that can inactivate specific antibodies, like those directed to the A-protein, a T-cell-independent (TI) mitogen, which was suggested to induce the production of monoclonal antibodies and to non-specifically bind immunoglobulin and, subsequently, also complement, thus possibly compromising S. aureus phagocytosis [
      • Albus A.
      • Fournier J.M.
      • Wolz C.
      • Boutonnier A.
      • Ranke M.
      • Høiby N.
      • et al.
      Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.
      ,
      • Kobayashi S.D.
      • DeLeo F.R.
      Staphylococcus aureus protein A promotes immune suppression.
      ].

      2.2.4 Alginate: cause or consequence of the ineffective humoral response?

      Despite the reports by Lang et al. suggesting that induction of high affinity anti-LPS antibodies may help to prevent the P. aeruginosa phenotypic switch, no additional study has corroborated this suggestion, as far as it is known. On the other hand, P. aeruginosa alginate is well known to be a major component of the P. aeruginosa biofilm, helping in its maturation, architecture and stability, as well as helping to protect the bacteria against phagocytosis and clearance from the lungs, by acting like an oxygen-radical scavenger, and inducing damage in the surrounding tissues [
      • Song Z.
      • Wu H.
      • Ciofu O.
      • Kong K.F.
      • Høiby N.
      • Rygaard J.
      • et al.
      Pseudomonas aeruginosa alginate is refractory to Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection.
      ,
      • Ciofu O.
      • Tolker-Nielsen T.
      • Jensen P.Ø.
      • Wang H.
      • Høiby N.
      Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients.
      ]. The fact of many PMNs are found surrounding the P. aeruginosa biofilms, but few or no PMNs are seen inside the biofilm (Fig. 1, Fig. 2) is in accordance with the protective activity of alginate, as well as with the lytic action of P. aeruginosa rhamnolipids on PMNs [
      • Bjarnsholt T.
      • Jensen P.Ø.
      • Fiandaca M.J.
      • Pedersen J.
      • Hansen C.R.
      • Andersen C.B.
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ,
      • Høiby N.
      • Ciofu O.
      • Bjarnsholt T.
      Pseudomonas aeruginosa biofilms in cystic fibrosis.
      ,
      • Jensen P.Ø.
      • Bjarnsholt T.
      • Phipps R.
      • Rasmussen T.B.
      • Calum H.
      • Christoffersen L.
      • et al.
      Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa.
      ].
      A pronounced antibody response against alginate is produced by CF patients (Fig. 1) [
      • Pedersen S.S.
      • Kharazmi A.
      • Espersen F.
      • Høiby N.
      Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response.
      ,
      • Pedersen S.S.
      • Møller H.
      • Espersen F.
      • Sørensen C.H.
      • Jensen T.
      • Høiby N.
      Mucosal immunity to Pseudomonas aeruginosa alginate in cystic fibrosis.
      ,
      • Craig A.
      • Mai J.
      • Cai S.
      • Jeyaseelan S.
      Neutrophil recruitment to the lungs during bacterial pneumonia.
      ], but opsonic quality has shown to be significantly declined in longitudinal analyses. Patients who passed age 12 free of P. aeruginosa colonization developed chronic P. aeruginosa lung infection later, suggesting that naturally occurring antibodies do not protect CF patients from mucoid P. aeruginosa infection, and opsonic quality of serum antibodies deteriorates as infection becomes established [
      • Tosi M.F.
      • Zakem-Cloud H.
      • Demko C.A.
      • Schreiber J.R.
      • Stern R.C.
      • Konstan M.W.
      • et al.
      Cross-sectional and longitudinal studies of naturally occurring antibodies to Pseudomonas aeruginosa in cystic fibrosis.
      ].
      Alginate is a polysaccharide antigen, and similar carbohydrate antigens are known to have poor immunogenicity due to the T-cell-independent (TI) nature [
      • Weintraub A.
      Immunology of bacterial polysaccharide antigens.
      ]. TI antigens induce an immunological memory characterized by high avidity antibodies, however they lack antibody production after new challenge with the antigen [
      • Mazmanian S.K.
      • Kasper D.L.
      The love–hate relationship between bacterial polysaccharides and the host immune system.
      ]. It has been shown that alginate is not able to fix complement and, therefore, cannot cause neutrophil chemotaxis by itself, but has the ability to enhance neutrophil-mediated oxidative burst [
      • Pedersen S.S.
      • Kharazmi A.
      • Espersen F.
      • Høiby N.
      Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response.
      ]. One report [
      • Meluleni G.J.
      • Grout M.
      • Evans D.J.
      • Pier G.B.
      Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic fibrosis patients.
      ] suggested that the opsonic activity of specific anti-alginate antibodies against mucoid P. aeruginosa in biofilms is related to their ability to deposit complement C3 fragments next to the bacterial surface, but sufficiently exposed so they can bind to complement receptors on the phagocytes, since alginate-specific opsonins were shown to deposit significantly more C3 fragments on alginate than non-alginate-specific opsonins. The authors still suggest that the alginate biofilm layer prevent complement receptors on the phagocytes to bind C3 fragments deposited by non-alginate-specific antibodies on other antigens, since biofilms treated with alginate lyase improved the opsonic killing mediated by non-alginate-specific antibodies in 12 from 16 P. aeruginosa chronically infected patients.
      Other reason for the difficulty to efficiently opsonize alginate-producing P. aeruginosa may be the predominance of the IgG2 response, since this IgG subclass responds to polysaccharide antigens and has poor opsonic quality [
      • Sigal L.H.
      Basic science for the clinician 58: IgG subclasses.
      ]. IgG competition with IgA by the alginate epitope may also play a role, since both serum and secretory IgA were shown to respond specifically when directed against purified P. aeruginosa alginate [
      • Pedersen S.S.
      • Kharazmi A.
      • Espersen F.
      • Høiby N.
      Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response.
      ,
      • Pedersen S.S.
      • Møller H.
      • Espersen F.
      • Sørensen C.H.
      • Jensen T.
      • Høiby N.
      Mucosal immunity to Pseudomonas aeruginosa alginate in cystic fibrosis.
      ,
      • Aanaes K.
      • Johansen H.K.
      • Poulsen S.S.
      • Pressler T.
      • Buchwald C.
      • Høiby N.
      Secretory IgA as a diagnostic tool for Pseudomonas aeruginosa respiratory colonization.
      ]. IgA was suggested to block alginate opsonization by IgG and, due to its poor opsonic capacity, to contribute to poor phagocytosis [
      • Pedersen S.S.
      • Espersen F.
      • Høiby N.
      • Jensen T.
      Immunoglobulin A and immunoglobulin G antibody responses to alginates from Pseudomonas aeruginosa in patients with cystic fibrosis.
      ]. Furthermore, we have shown that IgG avidity against alginate does not significantly increase with the progress of chronic infection, possibly playing a role in the difficulty to mediate efficient phagocytosis against this antigen to clear P. aeruginosa infection [
      • Mauch R.M.
      • Nørregard L.L.
      • Jensen P.Ø.
      • Levy C.E.
      • Høiby N.
      76 avidity of IgG antibodies and opsonic quality of serum samples against Pseudomonas aeruginosa during chronic lung infection in cystic fibrosis.
      ]. Previous studies [
      • Pier G.B.
      • DesJardin D.
      • Grout M.
      • Garner C.
      • Bennett S.E.
      • Pekoe G.
      • et al.
      Human immune response to Pseudomonas aeruginosa mucoid exopolysaccharide (alginate) vaccine.
      ,
      • Theilacker C.
      • Coleman F.T.
      • Mueschenborn S.
      • Llosa N.
      • Grout M.
      • Pier G.B.
      Construction and characterization of a Pseudomonas Aeruginosa mucoid exopolysaccharide-alginate conjugate vaccine.
      ] reported that alginate-based vaccines elicited opsonic antibodies in human, mice and rabbit even in the presence of preexisting non-opsonic antibodies [
      • Theilacker C.
      • Coleman F.T.
      • Mueschenborn S.
      • Llosa N.
      • Grout M.
      • Pier G.B.
      Construction and characterization of a Pseudomonas Aeruginosa mucoid exopolysaccharide-alginate conjugate vaccine.
      ]. Antibodies elicited by two vaccines enhanced deposition of complement onto mucoid P. aeruginosa cells and mediated opsonic killing of heterologous P. aeruginosa strains expressing different alginates [
      • Pier G.B.
      • DesJardin D.
      • Grout M.
      • Garner C.
      • Bennett S.E.
      • Pekoe G.
      • et al.
      Human immune response to Pseudomonas aeruginosa mucoid exopolysaccharide (alginate) vaccine.
      ]. However, no studies have so far addressed avidity changes against alginates during the course of infection.

      3. Concluding remarks

      The findings of immune dysregulation have strongly contributed in understanding CF inflammation mechanisms and directed researches of new therapeutic strategies for prevention and treatment. Inflammatory responses are designed for acute infection, aiming a fast clearance of the corresponding pathogen. In a chronic infection scenario, like CF, with repeated exposure to the pathogen, several and highly detrimental collateral damages are worrisome. To date, treatment regimens have focused on controlling the pro-inflammatory response in order to reduce the NF-kB signaling and consequently the neutrophilic influx in the airways. A therapeutic alternative would be to upregulate the anti-inflammatory mechanisms, thus directing the immune response to a protective phenotype. Therapies targeting the molecular and submolecular mechanisms and of biofilm formation will likely have a key role in clearing infection, helping to halt the perpetual neutrophilic response.
      The role of adaptive immune response also needs to be better exploited, especially regarding to the humoral response. Despite B-cells being directly activated by invading organisms, and CF chronic lung infections recruiting an elevated and apparently not effective antibody response, very few studies addressing antibody function in CF were published in the last 20 years, and B-cell features in CF has been reported only once, as far as it is known. Therefore, this field remains mostly unknown. Such antibody response is not inefficient by chance, and the underlying mechanisms, such as avidity/affinity maturation, class switch, memory formation and cytokine synthesis must be better elucidated.
      The discovery of new disease mechanisms and development of new therapies can create windows of opportunity to reduce the impact of inflammation, consequently reducing the decline in lung function in CF.

      Conflict of interests

      On behalf all authors, the corresponding author states that there are no competing interests.

      References

        • Stoltz D.
        • Meyerholz D.K.
        • Welsh M.J.
        Origins of cystic fibrosis lung disease.
        NEJM. 2015; 372: 351-362https://doi.org/10.1056/NEJMra1300109
        • Folkesson A.
        • Jelsbak L.
        • Yang L.
        • Johansen H.K.
        • Ciofu O.
        • Høiby N.
        • et al.
        Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective.
        Nat Rev Microbiol. 2012; 10: 841-851https://doi.org/10.1038/nrmicro2907
        • Moradali M.F.
        • Ghods S.
        • Rehm B.H.A.
        Pseudomonas aeruginosa lifestyle: a paradigm for adaptation, survival, and persistence.
        Front Cell Infect Microbiol. 2017; 7: 1-29https://doi.org/10.3389/fcimb.2017.00039
        • Abou Alaiwa M.H.
        • Reznikov L.R.
        • Gansemer N.D.
        • Sheets K.A.
        • Horswill A.R.
        • Stoltz D.A.
        • et al.
        pH modulates the activity and synergism of the airway surface liquid antimicrobials β-defensin-3 and LL-37.
        Proc Natl Acad Sci. 2014; 111: 18703-18708https://doi.org/10.1073/pnas.1422091112
        • Berkebile A.R.
        • McCray P.B.
        Effects of airway surface liquid pH on host defense in cystic fibrosis.
        Int J Biochem Cell Biol. 2014; 52: 124-129https://doi.org/10.1016/j.biocel.2014.02.009.Effects
        • Yonker L.M.
        • Cigana C.
        • Hurley B.P.
        • Bragonzi A.
        Host-pathogen interplay in the respiratory environment of cystic fibrosis.
        J Cyst Fibros. 2015; 14: 1-9https://doi.org/10.1016/j.jcf.2015.02.008
        • Ziady A.G.
        • Hansen J.
        Redox balance in cystic fibrosis.
        Int J Biochem Cell Biol. 2014; 52: 113-123https://doi.org/10.1016/j.biocel.2014.03.006
        • Parker D.
        • Cohen T.S.
        • Alhede M.
        • Harfenist B.S.
        • Martin F.J.
        • Prince A.
        Induction of type I interferon signaling by Pseudomonas aeruginosa is diminished in cystic fibrosis epithelial cells.
        Am J Respir Cell Mol Biol. 2012; 46: 6-13https://doi.org/10.1165/rcmb.2011-0080OC
        • Twigg M.S.
        • Brockbank S.
        • Lowry P.
        • FitzGerald S.P.
        • Taggart C.
        • Weldon S.
        The role of serine proteases and antiproteases in the cystic fibrosis lung.
        Mediat Inflamm. 2015; 2015: 1-10https://doi.org/10.1155/2015/293053
        • McCaslin C.A.
        • Petrusca D.N.
        • Poirier C.
        • Serban K.A.
        • Anderson G.G.
        • Petrache I.
        Impact of alginate-producing Pseudomonas aeruginosa on alveolar macrophage apoptotic cell clearance.
        J Cyst Fibros. 2015; 14: 70-77https://doi.org/10.1016/j.jcf.2014.06.009
        • Bruscia E.M.
        • Bonfield T.L.
        Cystic fibrosis lung immunity: the role of the macrophage.
        J Innate Immun. 2016; 8: 550-563https://doi.org/10.1159/000446825
        • Moss R.B.
        • Hsu Y.P.
        • Olds L.
        Cytokine dysregulation in activated cystic fibrosis (CF) peripheral lymphocytes.
        Clin Exp Immunol. 2000; 120: 518-525https://doi.org/10.1046/j.1365-2249.2000.01232.x
        • Casaulta C.
        • Schöni M.H.
        • Weichel M.
        • Crameri R.
        • Jutel M.
        • Daigle I.
        • et al.
        IL-10 controls aspergillus fumigatus- and Pseudomonas aeruginosa-specific T-cell response in cystic fibrosis.
        Pediatr Res. 2003; 53: 313-319https://doi.org/10.1203/01.PDR.0000047528.79014.CF
        • Hartl D.
        • Griese M.
        • Kappler M.
        • Zissel G.
        • Reinhardt D.
        • Rebhan C.
        • et al.
        Pulmonary TH2 response in Pseudomonas aeruginosa-infected patients with cystic fibrosis.
        J Allergy Clin Immunol. 2006; 117: 204-211https://doi.org/10.1016/j.jaci.2005.09.023
        • Moser C.
        • Jensen P.O.
        • Kobayashi O.
        • Hougen H.P.
        • Song Z.
        • Rygaard J.
        • et al.
        Improved outcome of chronic Pseudomonas aeruginosa lung infection is associated with induction of a Th1-dominated cytokine response.
        Clin Exp Immunol. 2002; 127: 206-213https://doi.org/10.1046/j.1365-2249.2002.01731.x
        • Johansen H.K.
        • Hougen H.P.
        • Rygaard J.
        • Høiby N.
        Interferon-gamma (IFN-gamma) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats.
        Clin Exp Immunol. 1996; 103: 212-218
        • Moser C.
        • Pedersen H.T.
        • Lerche C.J.
        • Kolpen M.
        • Line L.
        • Thomsen K.
        • et al.
        Biofilms and host response: helpful or harmful?.
        APMIS. 2017; 125: 320-338https://doi.org/10.1111/apm.12674
        • Tiringer K.
        • Treis A.
        • Fucik P.
        • Gona M.
        • Gruber S.
        • Renner S.
        • et al.
        A Th17- and Th2-skewed cytokine profile in cystic fibrosis lungs represents a potential risk factor for Pseudomonas aeruginosa infection.
        Am J Respir Crit Care Med. 2013;
        • Kushwah R.
        • Gagnon S.
        • Sweezey N.B.
        Intrinsic predisposition of naïve cystic fibrosis T cells to differentiate towards a Th17 phenotype.
        Respir Res. 2013; 14: 138https://doi.org/10.1186/1465-9921-14-138
        • Bayes H.K.
        • Bicknell S.
        • MacGregor G.
        • Evans T.J.
        T helper cell subsets specific for Pseudomonas aeruginosa in healthy individuals and patients with cystic fibrosis.
        PLoS One. 2014; 9https://doi.org/10.1371/journal.pone.0090263
        • Decraene A.
        • Willems-Widyastuti A.
        • Kasran A.
        • De Boeck K.
        • Bullens D.M.
        • Dupont L.J.
        Elevated expression of both mRNA and protein levels of IL-17A in sputum of stable cystic fibrosis patients.
        Respir Res. 2010; 11: 177-184https://doi.org/10.1186/1465-9921-11-177
        • Høiby N.
        • Flensborg E.W.
        • Beck B.
        • Friis B.
        • Jacobsen S.V.
        • Jacobsen L.
        Pseudomonas aeruginosa infection in cystic fibrosis. Diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis.
        Scand J Respir Dis. 1977; 58: 65-79
        • Mauch R.M.
        • Rossi C.L.
        • Ribeiro J.D.
        • Ribeiro A.F.
        • Nolasco da Silva M.T.
        • Levy C.E.
        Assessment of IgG antibodies to Pseudomonas aeruginosa in patients with cystic fibrosis by an enzyme-linked immunosorbent assay (ELISA).
        Diagn Pathol. 2014; 9: 158-165https://doi.org/10.1186/s13000-014-0158-z
        • Mauch R.M.
        • Levy C.E.
        Serum antibodies to Pseudomonas aeruginosa in cystic fibrosis as a diagnostic tool: a systematic review.
        J Cyst Fibros. 2014; 13: 499-507https://doi.org/10.1016/j.jcf.2014.01.005
        • Brett M.M.
        • Ghoneim A.T.
        • Littlewood J.M.
        Prediction and diagnosis of early Pseudomonas aeruginosa infection in cystic fibrosis: a follow-up study [published erratum appears in J Clin Microbiol 1989 Jan 27 (1):230].
        J Clin Microbiol. 1988; 26: 1565-1570
        • West S.E.H.
        • Zeng L.
        • Lee B.L.
        • Kosorok M.R.
        • Laxova A.
        • Rock M.J.
        • et al.
        Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors.
        JAMA. 2002; 287: 2958-2967
        • Anstead M.
        • Heltshe S.L.
        • Khan U.
        • Barbieri J.T.
        • Langkamp M.
        • Döring G.
        • et al.
        Pseudomonas aeruginosa serology and risk for re-isolation in the EPIC trial.
        J Cyst Fibros. 2013; 12: 147-153https://doi.org/10.1016/j.jcf.2012.08.001
        • Lebien T.W.
        • Tedder T.F.
        B lymphocytes : how they develop and function.
        Am Soc Hematol. 2008; 112: 1570-1580https://doi.org/10.1182/blood-2008-02-078071
        • Hoffman W.
        • Lakkis F.G.
        • Chalasani G.
        B cells, antibodies, and more.
        Clin J Am Soc Nephrol. 2016; 11: 137-154https://doi.org/10.2215/CJN.09430915
        • Sorensen R.U.
        • Ruuskanen O.
        • Miller K.
        • Stern R.C.
        B-lymphocyte function in cystic fibrosis.
        Eur J Respir Dis. 1983; 64: 524-533
        • Neill D.R.
        • Saint G.L.
        • Bricio-Moreno L.
        • Fothergill J.L.
        • Southern K.W.
        • Winstanley C.
        • et al.
        The B lymphocyte differentiation factor (BAFF) is expressed in the airways of children with CF and in lungs of mice infected with Pseudomonas aeruginosa.
        PLoS One. 2014; 9: 3-9https://doi.org/10.1371/journal.pone.0095892
        • McFarlane H.
        • Holzel A.
        • Brenchley P.
        • Allan J.D.
        • Wallwork J.C.
        • Singer B.E.
        • et al.
        Immune complexes in cystic fibrosis.
        Br Med J. 1975; 1: 423-428
        • Schiøtz P.O.
        • Nietsen H.
        • Høiby N.
        • Glikmann G.
        • Svehag S.E.
        Immune complexes in the sputum of patients with cystic fibrosis suffering from chronic Pseudomonas aeruginosa lung infection.
        APMIS. 1978; 86C: 37-40https://doi.org/10.1111/j.1699-0463.1978.tb02555.x
        • Hornick D.B.
        • Fick Jr., R.B.
        The immunoglobulin G subclass composition of immune complexes in cystic fibrosis.
        J Clin Invest. 1990; 86: 1285-1292
        • Kronborg G.
        • Fomsgaard A.
        • Galanos C.
        • Freudenberg M.
        • Høiby N.
        Antibody responses to lipid A, core, and O sugars of the Pseudomonas aeruginosa lipopolysaccharide in chronically infected cystic fibrosis patients.
        J Clin Microbiol. 1992; 30: 1848-1855
        • Moss R.B.
        • Hsu Y.P.
        • Lewiston N.J.
        • Curd J.G.
        • Milgrom H.
        • Hart S.
        • et al.
        Association of systemic immune complexes, complement activation, and antibodies to Pseudomonas aeruginosa lipopolysaccharide and exotoxin A with mortality in cystic fibrosis.
        Am Rev Respir Dis. 1986; 133: 648-652
        • Moss R.B.
        • Lewiston N.J.
        Immune complexes and humoral response to Pseudomonas aeruginosa in cystic fibrosis.
        Am Rev Respir Dis. 1980; 121: 23-29
        • Church J.A.
        • Jordan S.C.
        • Keens T.G.
        • Wang C.I.
        Circulating immune complexes in patients with cystic fibrosis.
        Chest. 1981; 80: 405-411https://doi.org/10.1378/chest.80.4.405
        • Hodson M.E.
        • Beldon I.
        • Batten J.C.
        Circulating immune complexes in patients with cystic fibrosis.
        Clin Allergy. 1985; 15: 363-370https://doi.org/10.1111/j.1365-2222.1985.tb03004.x
        • Van Bever H.P.
        • Gigase P.L.
        • De Clerck L.S.
        • Bridts C.H.
        • Franckx H.
        • Stevens W.J.
        Immune complexes and Pseudomonas aeruginosa antibodies in cystic fibrosis.
        Arch Dis Child. 1988; 63: 1222-1228https://doi.org/10.1136/adc.63.10.1222
        • Lagacé J.
        • Mercier J.
        • Frechette M.
        • Fournier D.
        • Dubreuil M.
        • Lamarre A.
        • et al.
        Circulating immune complexes, antibodies to Pseudomonas aeruginosa, and pulmonary status in cystic fibrosis.
        J Clin Lab Immunol. 1989; 30: 7-11
        • Disis M.L.
        • McDonald T.L.
        • Colombo J.L.
        • Kobayashi R.H.
        • Angle C.R.
        • Murray S.
        Circulating immune complexes in cystic fibrosis and their correlation to clinical parameters.
        Pediatr Res. 1986; 20: 385-390https://doi.org/10.1203/00006450-198605000-00002
        • Wisnieski J.J.
        • Todd E.W.
        • Fuller R.K.
        • Jones P.K.
        • Dearborn D.G.
        • Boat T.F.
        • et al.
        Immune complexes and complement abnormalities in patients with cystic fibrosis. Increased mortality associated with circulating immune complexes and decreased function of the alternative complement pathway.
        Am Rev Respir Dis. 1985; 132: 770-776
        • Berdischewsky M.
        • Pollack M.
        • Young L.S.
        • Chia D.
        • Osher A.B.
        • Barnett E.V.
        Circulating immune complexes in cystic.
        Pediatr Res. 1980; 14: 830-833
        • Pitcher-Wilmott R.W.
        • Levinsky R.J.
        • Matthew D.J.
        Circulating soluble immune complexes containing pseudomonas antigens in cystic fibrosis.
        Arch Dis Child. 1982; 57: 577-581https://doi.org/10.1136/adc.57.8.577
        • Döring G.
        • Albus A.
        • Høiby N.
        Immunologic aspects of cystic fibrosis.
        Chest. 1988; 94: 109S-115S
        • Czuprynski C.J.
        Opsonization and phagocytosis.
        in: Encycl. ref. immunotoxicol. Springer-Verlag, Berlin/Heidelberg2005: 488-489https://doi.org/10.1007/3–540-27806-0_1113
        • Høiby N.
        • Schiøtz P.O.
        Immune complex mediated tissue damage in the lungs of cystic fibrosis patients with chronic Pseudomonas aeruginosa infection.
        Acta Paediatr. 1982; : 63-73
        • Thomassen M.J.
        • Demko C.A.
        Serum bactericidal effect on Pseudomonas aeruginosa isolates from cystic fibrosis patients.
        Infect Immun. 1981; 33: 512-518
        • Guttman R.M.
        • Waisbren B.A.
        Bacterial blocking activity of specific IgG in chronic Pseudomonas aeruginosa infection.
        Clin Exp Immunol. 1975; 19: 121-130
        • LeBlanc C.M.A.
        • Bortolussi R.
        • Issekutz A.C.
        • Gillespie T.
        Opsonization of mucoid and non-mucoid Pseudomonas aeruginosa by serum from patients with cystic fibrosis assessed by a chemiluminescence assay.
        Clin Investig Med. 1982; 5: 125-128
        • Moss R.B.
        • Hsu Y.P.
        • Sullivan M.M.
        • Lewiston N.J.
        Altered antibody isotype in cystic fibrosis: possible role in opsonic deficiency.
        Pediatr Res. 1986; 20: 453-459
        • Thomassen M.J.
        • Boxerbaum B.
        • Demko C.A.
        • Kuchenbrod P.J.
        • Dearborn D.G.
        • Wood R.E.
        Inhibitory effect of cystic fibrosis serum on Pseudomonas phagocytosis by rabbit and human alveolar macrophages.
        Pediatr Res. 1979; 13: 1085-1088
        • Thomassen M.J.
        • Demko C.A.
        • Wood R.E.
        • Tandler B.
        • Dearborn D.G.
        • Boxerbaum B.
        • et al.
        Ultrastructure and function of alveolar macrophages from cystic fibrosis patients.
        Pediatr Res. 1980; 14: 715-721https://doi.org/10.1203/00006450-198005000-00003
        • Pier G.B.
        • Saunders M.J.
        • Ames P.
        • Edwards M.S.
        • Auerbach H.
        • Goldfarb J.
        • et al.
        Opsonophagocytic killing antibody to Pseudomonas aeruginosa mucoid exopolysaccharide in older noncolonized patients with cystic fibrosis.
        N Engl J Med. 1987; 317: 793-798
        • Moss R.B.
        The role of IgG subclass antibodies in chronic infection: the case of cystic fibrosis.
        N Engl Reg Allergy Proc. 1988; 9: 57-61
        • Pressler T.
        • Jensen E.T.
        • Espersen F.
        • Pedersen S.S.
        • Høiby N.
        High levels of complement-activation capacity in sera from patients with cystic fibrosis correlate with high levels of IgG3 antibodies to Pseudomonas aeruginosa antigens and poor lung function.
        Pediatr Pulmonol. 1995; 20: 71-77https://doi.org/10.1002/ppul.1950200204
        • Eichler I.
        • Joris L.
        • Hsu Y.P.
        • Van Wye J.
        • Bram R.
        • Moss R.
        Nonopsonic antibodies in cystic fibrosis. Pseudomonas aeruginosa lipopolysaccharide-specific immunoglobulin G antibodies from infected patient sera inhibit neutrophil oxidative responses.
        J Clin Invest. 1989; 84: 1794-1804https://doi.org/10.1172/JCI114364
        • Thomsen K.
        • Christophersen L.
        • Bjarnsholt T.
        • Jensen P.
        • Moser C.
        • Høiby N.
        Anti-Pseudomonas aeruginosa IgY antibodies promote specific bacterial aggregation and internalization in polymorphonuclear neutrophils.
        Infect Immun. 2013; 83: 2686-2693https://doi.org/10.1128/IAI.02970-14
        • Thomsen K.
        • Christophersen L.
        • Jensen P.Ø.
        • Bjarnsholt T.
        • Moser C.
        • Høiby N.
        Anti-Pseudomonas aeruginosa IgY antibodies promote bacterial opsonization and augment the phagocytic activity of polymorphonuclear neutrophils.
        Hum Vaccines Immunother. 2016; 12: 1690-1699https://doi.org/10.1080/21645515.2016.1145848
        • Bjarnsholt T.
        • Jensen P.Ø.
        • Fiandaca M.J.
        • Pedersen J.
        • Hansen C.R.
        • Andersen C.B.
        • et al.
        Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
        Pediatr Pulmonol. 2009; 44: 547-558https://doi.org/10.1002/ppul.21011
        • Holland S.M.
        Chronic granulomatous disease.
        Clin Rev Allergy Immunol. 2010; 38: 3-10https://doi.org/10.1007/s12016-009-8136-z
        • Kolpen M.
        • Mousavi N.
        • Sams T.
        • Bjarnsholt T.
        • Ciofu O.
        • Moser C.
        • et al.
        Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment.
        Int J Antimicrob Agents. 2016; 47: 163-167https://doi.org/10.1016/j.ijantimicag.2015.12.005
        • Gutiérrez J.
        • Maroto C.
        Are IgG antibody avidity assays useful in the diagnosis of infectious diseases? A review.
        Microbios. 1996; 87: 113-121
        • Dimitrov J.D.
        • Planchais C.
        • Roumenina L.T.
        • Vassilev T.L.
        • Kaveri S.V.
        • Lacroix-Desmazes S.
        Antibody polyreactivity in health and disease: statu variabilis.
        J Immunol. 2013; 191: 993-999https://doi.org/10.4049/jimmunol.1300880
        • Fick R.B.
        • Naegel G.P.
        • Matthay R.A.
        • Reynolds H.Y.
        Cystic fibrosis Pseudomonas opsonins. Inhibitory nature in an in vitro phagocytic assay.
        J Clin Invest. 1981; 68: 899-914
        • Bruderer U.
        • Cryz S.J.
        • Schaad U.B.
        • Deusinger M.
        • Que J.U.
        • Lang A.B.
        Affinity constants of naturally acquired and vaccine-induced anti-Pseudomonas aeruginosa antibodies in healthy adults and cystic fibrosis patients.
        J Infect Dis. 1992; 166: 344-349
        • Torensma R.
        • Fluit A.C.
        • Verhoef J.
        Reactivity of monoclonal antibodies to Pseudomonas aeruginosa isolates from hospitalized adults and patients with cystic fibrosis.
        Clin Infect Dis. 1994; 19: 11-14
        • Lang A.B.
        • Schaad U.B.
        • Rüdeberg A.
        • Wedgwood J.
        • Que J.U.
        • Fürer E.
        • et al.
        Effect of high-affinity anti-Pseudomonas aeruginosa lipopolysaccharide antibodies induced by immunization on the rate of Pseudomonas aeruginosa infection in patients with cystic fibrosis.
        J Pediatr. 1995; 127: 711-717
        • Polanec J.
        • Patzer J.
        • Grzybowski J.
        • Strukelj M.
        • Pavelic Z.P.
        Amount and avidity of IgG antibodies to Pseudomonas aeruginosa exotoxin a antigen in cystic fibrosis patients.
        Pathol Oncol Res. 1997; 3: 26-29
        • Brauner A.
        • Cryz S.J.
        • Granström M.
        • Hanson H.S.
        • Löfstrand L.
        • Strandvik B.
        • et al.
        Immunoglobulin G antibodies to Pseudomonas aeruginosa lipopolysaccharides and exotoxin A in patients with cystic fibrosis or bacteremia.
        Eur J Clin Microbiol Infect Dis. 1993; 12: 430-436
        • Ciofu O.
        • Petersen T.D.
        • Jensen P.Ø.
        • Høiby N.
        Avidity of anti-P. aeruginosa antibodies during chronic infection in patients with cystic fibrosis.
        Thorax. 1999; 54: 141-144https://doi.org/10.1136/thx.54.2.141
        • Albus A.
        • Fournier J.M.
        • Wolz C.
        • Boutonnier A.
        • Ranke M.
        • Høiby N.
        • et al.
        Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.
        J Clin Microbiol. 1988; 26: 2505-2509
        • Kobayashi S.D.
        • DeLeo F.R.
        Staphylococcus aureus protein A promotes immune suppression.
        MBio. 2013; 4: e00764-13https://doi.org/10.1128/mBio.00764-13
        • Song Z.
        • Wu H.
        • Ciofu O.
        • Kong K.F.
        • Høiby N.
        • Rygaard J.
        • et al.
        Pseudomonas aeruginosa alginate is refractory to Th1 immune response and impedes host immune clearance in a mouse model of acute lung infection.
        J Med Microbiol. 2003; 52: 731-740https://doi.org/10.1099/jmm.0.05122-0
        • Ciofu O.
        • Tolker-Nielsen T.
        • Jensen P.Ø.
        • Wang H.
        • Høiby N.
        Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients.
        Adv Drug Deliv Rev. 2015; 85: 7-23https://doi.org/10.1016/j.addr.2014.11.017
        • Høiby N.
        • Ciofu O.
        • Bjarnsholt T.
        Pseudomonas aeruginosa biofilms in cystic fibrosis.
        Future Microbiol. 2010; 5: 1663-1674
        • Jensen P.Ø.
        • Bjarnsholt T.
        • Phipps R.
        • Rasmussen T.B.
        • Calum H.
        • Christoffersen L.
        • et al.
        Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa.
        Microbiology. 2007; 153: 1329-1338https://doi.org/10.1099/mic.0.2006/003863-0
        • Pedersen S.S.
        • Kharazmi A.
        • Espersen F.
        • Høiby N.
        Pseudomonas aeruginosa alginate in cystic fibrosis sputum and the inflammatory response.
        Infect Immun. 1990; 58: 3363-3368
        • Pedersen S.S.
        • Møller H.
        • Espersen F.
        • Sørensen C.H.
        • Jensen T.
        • Høiby N.
        Mucosal immunity to Pseudomonas aeruginosa alginate in cystic fibrosis.
        APMIS. 1992; 100: 326-334
        • Craig A.
        • Mai J.
        • Cai S.
        • Jeyaseelan S.
        Neutrophil recruitment to the lungs during bacterial pneumonia.
        Infect Immun. 2009; 77: 568-575https://doi.org/10.1128/IAI.00832-08
        • Tosi M.F.
        • Zakem-Cloud H.
        • Demko C.A.
        • Schreiber J.R.
        • Stern R.C.
        • Konstan M.W.
        • et al.
        Cross-sectional and longitudinal studies of naturally occurring antibodies to Pseudomonas aeruginosa in cystic fibrosis.
        J Infect Dis. 1995; 172: 453-461
        • Weintraub A.
        Immunology of bacterial polysaccharide antigens.
        Carbohydr Res. 2003; 338: 2539-2547https://doi.org/10.1016/j.carres.2003.07.008
        • Mazmanian S.K.
        • Kasper D.L.
        The love–hate relationship between bacterial polysaccharides and the host immune system.
        Nat Rev Immunol. 2006; 6: 849-858https://doi.org/10.1038/nri1956
        • Meluleni G.J.
        • Grout M.
        • Evans D.J.
        • Pier G.B.
        Mucoid Pseudomonas aeruginosa growing in a biofilm in vitro are killed by opsonic antibodies to the mucoid exopolysaccharide capsule but not by antibodies produced during chronic lung infection in cystic fibrosis patients.
        J Immunol. 1995; 155: 2029-2038https://doi.org/10.4049/jimmunol.173.5.3320
        • Sigal L.H.
        Basic science for the clinician 58: IgG subclasses.
        J Clin Rheumatol. 2012; 18: 316-318https://doi.org/10.1097/RHU.0b013e318269446b
        • Aanaes K.
        • Johansen H.K.
        • Poulsen S.S.
        • Pressler T.
        • Buchwald C.
        • Høiby N.
        Secretory IgA as a diagnostic tool for Pseudomonas aeruginosa respiratory colonization.
        J Cyst Fibros. 2013; 12: 81-87https://doi.org/10.1016/j.jcf.2012.07.001
        • Pedersen S.S.
        • Espersen F.
        • Høiby N.
        • Jensen T.
        Immunoglobulin A and immunoglobulin G antibody responses to alginates from Pseudomonas aeruginosa in patients with cystic fibrosis.
        J Clin Microbiol. 1990; 28: 747-755
        • Mauch R.M.
        • Nørregard L.L.
        • Jensen P.Ø.
        • Levy C.E.
        • Høiby N.
        76 avidity of IgG antibodies and opsonic quality of serum samples against Pseudomonas aeruginosa during chronic lung infection in cystic fibrosis.
        J Cyst Fibros. 2017; 16: S83-4https://doi.org/10.1016/S1569-1993(17)30440-X
        • Pier G.B.
        • DesJardin D.
        • Grout M.
        • Garner C.
        • Bennett S.E.
        • Pekoe G.
        • et al.
        Human immune response to Pseudomonas aeruginosa mucoid exopolysaccharide (alginate) vaccine.
        Infect Immun. 1994; 62: 3972-3979
        • Theilacker C.
        • Coleman F.T.
        • Mueschenborn S.
        • Llosa N.
        • Grout M.
        • Pier G.B.
        Construction and characterization of a Pseudomonas Aeruginosa mucoid exopolysaccharide-alginate conjugate vaccine.
        Infect Immun. 2003; 71: 3875-3884https://doi.org/10.1128/IAI.71.7.3875