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Current concepts of immune dysregulation in cystic fibrosis夽 N. Rieber, A. Hector, M. Carevic, D. Hartl ∗ CF Research Group, Department of Pediatrics I, University of Tübingen, Tübingen, Germany
a r t i c l e Article history: Available online xxx Keywords: Cystic fibrosis Immunity Innate Toll-like receptors Neutrophils
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a b s t r a c t Cystic fibrosis (CF) lung disease is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and is characterized by a perpetuated feedback loop of bacterial infection and inflammation. Both intrinsic (CFTR-dependent) and extrinsic (CFTR-independent) mechanisms contribute to the inflammatory phenotype of CF lung disease. Innate immune cells, initially recruited to combat bacterial pathogens, are acting in a dysregulated and non-resolving fashion in CF airways and cause harm to the host by releasing proteases and oxidants. Targeting harmful immune pathways, while preserving protective ones, remains the challenge for the future. This review highlights current concepts of innate immune dysregulation in CF lung disease. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. © 2014 Elsevier Ltd. All rights reserved.
1. Immunity in cystic fibrosis: more questions than answers Cystic fibrosis (CF) lung disease is characterized by a chronic and non-resolving activation of the innate immune system with release of chemokines and an infiltration of neutrophils into the airways. Neutrophil-derived oxidants and proteases cause harm to multiple cellular and humoral factors and have recently been shown to impair Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) functionality itself (Le Gars et al., 2013). Beyond neutrophils, the immune response in CF lungs is dysregulated at several levels, including impaired (i) ceramide homeostasis, (ii) apoptosis, (iii) autophagy, (iv) macrophage polarization and functionality, (v) Tcell responses (Th2/Th17 predominance) and other deviations as reviewed recently (Hartl et al., 2012). Research in innate immunity in CF is aimed at (i) understanding the key mechanisms driving this pro-inflammatory vicious circle, (ii) identifying biomarkers that predict the course of disease and (iii) targeting specific components of the innate immune system as future therapeutics. However, our precise understanding of innate immunity in CF is handicapped by several uncertainties in the field: 1. The chicken-and-egg issue: what comes first? Inflammation or bacterial infection in the early course of CF lung disease? Is there intrinsic CFTR-dependent inflammation also without any prior
夽 This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. ∗ Corresponding author. Tel.: +49 70712981460. E-mail address: [email protected] (D. Hartl).
infection or were our methods to detect infection in previous studies just not sensitive enough to assess microbial impact? Recent insights from culture-independent microbial detection techniques have to be taken into account when addressing this question (Rabin and Surette, 2012). Countries with CF newborn screening and bronchoalveolar lavage (BAL) studies have shed light on this relationship (Sly et al., 2013) and new animal models, primarily the CF pig (Rogers et al., 2008), will help to answer this question. 2. Inter-species differences: Basic immunology relies on murine models to dissect pathways that contribute to disease. However, both (i) the lung anatomy and (ii) the myeloid cell composition differ substantially between mouse and man. New animal models, namely the CF ferret and the CF pig, present major advantages to overcome these hurdles, but studies are required to comprehensively characterize their immune responses and to assess their potential as therapeutically useful model systems to target inflammation in CF lung disease. 3. Who’s bad? Are neutrophils, the most abundant inflammatory cell in CF airways, good or bad? If we deplete them, our first cellular shield against bacteria and fungi is lacking, but there is compelling evidence that neutrophil-derived products, mainly proteases, cause harm to the extracellular pulmonary matrix. Therefore, one might target neutrophil products, for instance proteases. However, these approaches showed limited success so far in clinical studies. As there are several functionally distinct phenotypes of neutrophils (Borregaard, 2010; Kolaczkowska and Kubes, 2013), one could target harmful subtypes while preserving protective ones. But which markers discriminate these subtypes? What is the role of myeloid-derived
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suppressor cells (MDSCs) that dampen overshooting T cell activation (Gabrilovich and Nagaraj, 2009)? What about macrophages? They are required as potent phagocytic cell type, clearing both pathogens and – maybe more important for advanced CF lung disease – apoptotic cells through efferocytosis (Vandivier et al., 2009). Macrophages can be subdivided into classical M1 and M2 macrophages, the latter being induced in a Th2-environment and driving fibrosis and extracellular matrix remodeling. Data on M1/M2 macrophage phenotypes in CF are controversial, so it’s too early to target one of them, at least from our point of view. Data on other innate immune cell types, such as dendritic cells, natural killer (NK) cells, NKT cells and innate lymphoid cells (ILC) are scarce in the field of CF and additional studies are required to define their potential roles and CF disease relevance. 4. Diagnostic consequences: Which biomarkers could indicate patients who are prone to a rapid loss of lung function and should be monitored with greater caution? A recent study shows that free elastase in BAL fluid may represent such a predictive biomarker (Sly et al., 2013). However, BAL is hardly implementable in many clinical CF routines. Elastase in sputum could serve as an alternative, but is not – or hardly – feasible in infants below five years of age and requires standardized pre-analytical processing for reproducible biomarker analyses. 5. Therapeutic consequences: The cycle of inflammatory cell recruitment and unopposed immune cell activation causes tissue damage and leads to irreversible loss of lung function. Proteases and reactive oxygen species (ROS), released by infiltrating myeloid cells, are two key components in this cycle. Consequently, using anti-proteases and/or anti-oxidants are two mechanistic anti-inflammatory approaches in CF lung disease. However, several attempts to target these pathways were only partially successful, presumably due to (i) too low drug concentrations deposited at the pulmonary target site and (ii) inclusion of older CF patients with advanced lung disease characterized by irreversibly-remodeled and – damaged lung structure (Griese et al., 2008, Gaggar et al., 2011). What do we learn from this? How early do we have to start targeting proteases and oxidants to achieve clinical success? What about the compartmentalization of candidate drugs? It is critical that compounds reach their target in the midst of the acidic, oxidative, hyperviscous and hyperproteolytic micro-environment of the CF airway lumen. Are small-molecule compounds superior to natural protease inhibitors or do they show severe side effects as they also inhibit intracellular proteases that are involved in bacterial killing? Accordingly, it would make sense to engineer cell-impermeable compounds. All these issues have to be considered for new drug developments in CF lung disease. In the section below, we summarize and discuss selected recent findings in the field of innate immunity in CF lung disease. 2. Immunity in cystic fibrosis: recent findings 2.1. CFTR in myeloid cells Previous studies provided convincing evidence that human neutrophils express the CFTR protein, localized in the membrane of secretory vesicles (Painter et al., 2006). Optimal microbicidal activity of neutrophils relies on the generation of toxic agents such as hypochlorous acid (HOCl) within phagosomes (Hampton et al., 1998). HOCl formation, in turn, requires chloride ion transported from the cytoplasm into phagosomes mediated by chloride channels (Nauseef, 2007). Studies demonstrated that neutrophils pretreated with a CFTR inhibitor or siRNA-mediated CFTR
knock-down in neutrophil-like HL-60 cells reduced Cl− transport into phagosomes and impaired killing of phagocytosed bacteria (Painter et al., 2006, 2010; Bonvillain et al., 2010). Therefore, CFTR seems to play a role in regulating antimicrobial neutrophil activities. Zhou et al. (2013) recently confirmed and extended these studies by showing that nearly all mature phagosomes of human peripheral blood neutrophils are CFTR positive. The authors further stably expressed enhanced green fluorescent protein (EGFP) together with wt-CFTR or dF508-CFTR, respectively, in a promyelocytic cell line, where EGFP-wt-CFTR associated with phagosomes. In contrast, significantly less dF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Notably, the CFTR corrector compound VRT-325 facilitated the recruitment of dF508-CFTR to phagosomes. When viewed in combination, these data demonstrated the possibility of pharmacologic correction of impaired recruitment of mutant CFTR to the phagosome. This approach might enhance the chloride supply into the phagosomes of neutrophils in CF patients and increase their antimicrobial function (Zhou et al., 2013). However, promyelocytic cell lines, such as HL-60 cells or PLB-985 cells, used in the latter study, are only partially adequate models to study phagocytosis and other neutrophil functionalities, since their vesicle and granule compositions differ from primary human neutrophils and are therefore restricted as cellular model systems when recapitulating the physiological sequence of vesicle and granule membrane fusion events involved in phagocytosis by mature neutrophils. In contrast to patients with primary (inherited) immunodeficiencies in neutrophil function, such as chronic granulomatous disease, CF patients are not affected by recurrent invasive infections in general. Hence, the nature of neutrophil dysfunction in CF patients, has to be more subtle and is probably only of major disease relevance for (i) the extravascular pulmonary site of infection and for (ii) defined stages of host–pathogen interactions, an issue that, however, remains poorly understood. Recent bone marrow transplant studies corroborated the functional impact of myeloid Cftr in vivo (Su et al., 2011). Using conditional inactivation of Cftr in myeloid cells by means of a myeloid-targeted Cre-recombinase, Bonfield et al. demonstrated that the conditional “myeloid CF mice” displayed survival and inflammatory characteristics between wildtype and full genotypic Cftr−/− mice in P. aeruginosa infection models, depending on the stage of infection (Bonfield et al., 2012). In particular, the contribution of myeloid CFTR was most critical at later post-infection time points compared to the whole CFTR-deficient mice, supporting the notion that epithelial CFTR is involved in acute anti-bacterial host defense, while myeloid CFTR comes one step later when myeloid cells have entered the airways. Effects on other CF-associated phenotypes, such as epithelial ion transport, growth reduction or intestinal obstruction, as observed in the total absence of functional Cftr, were not detected in the myeloid-targeted conditional CF mouse model. In contrast to these studies, Oceandy et al. reported earlier in another CF mouse model that Cftr gene complementation in airway epithelium was sufficient to normalize pathogen clearance and inflammatory anomalies (Oceandy et al., 2002), suggesting that epithelial rather than myeloid CFTR is essential. The differing results may be due to the mouse models and the Cftr complementation/targeting methods used. Further comparative studies in mice and men are required to precisely define the contribution of myeloid CFTR to CF lung disease. 2.2. Myeloid-derived suppressor cells Myeloid-derived suppressor cells are innate immune cells generated in cancer and pro-inflammatory microenvironments (Gabrilovich and Nagaraj, 2009). These specialized innate immune
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Th17 suppression may regulate the immunopathogenesis and disease progression in P. aeruginosa infected CF patients. Importantly, P. aeruginosa downregulates its flagellin expression when exposed to airway secretions from CF patients with chronic lung infections (Wolfgang et al., 2004). In addition, flagellin detection by the immune system is also hindered in mucoid strains of P. aeruginosa or within biofilms. Therefore, the effect of P. aeruginosa on MDSCs may depend on several bacterial factors and is probably more complex. Moreover, the exact locations of MDSC–Th17 interactions (blood versus lamina propria versus lumen of the CF lung), the signaling dominance and disease relevance of MDSCs at various stages of CF lung disease remain to be investigated in future studies.
2.3. Toll-like receptors
Fig. 1. Pseudomonas – MDSC – T cell–host-defense circuit in the CF lung. Continuous Pseudomonas – phagocyte contacts result in the release of pro-inflammatory mediators that are capable of driving Th17 generation. Pseudomonas-derived flagellin induces MDSCs that suppress Th17 responses and affect the inflammatory CF circuit at two levels: first, CD4+ T cell proliferation is suppressed resulting in quantitatively less effector T cells. Second, IL-17 release by CD4+ T cells is dampened, leading in combination to a decrease of functional Th17 cells in CF. The pathophysiological consequences of this MDSC–Th17 interaction in the context of host–pathogen interactions and disease progression could be two-fold: on the one hand the reduction of bioactive IL-17 implies a decrease of neutrophil recruitment and phagocytic activity, favoring the survival of P. aeruginosa. On the other hand, MDSCs may dampen excessive Th17-neutrophil activation thereby preventing or limiting auto-destructive host tissue damage mediated through neutrophil-derived proteases.
cells are characterized by their capacity to suppress T-cell responses and thereby modulate adaptive immunity. Consequently, MDSCs are considered as a key intermediary in balancing innate and adaptive immune responses, particularly under chronic disease conditions (Gabrilovich and Nagaraj, 2009). We recently demonstrated that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa (Rieber et al., 2013). The percentage of MDSCs in the peripheral blood correlated indirectly with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was mimicked by purified flagellin protein and significantly reduced using flagellin-deficient P. aeruginosa bacteria. Functionally, MDSCs efficiently suppressed polyclonal T cell proliferation and modulated Th17 responses. These studies demonstrated that flagellin induces the generation of MDSCs and suggest that P. aeruginosa utilizes this mechanism to undermine T cell-mediated host defense in CF. Importantly, recent lines of evidence indicate that T helper 17 (Th17) cells and the IL-17 axis-related cytokines IL-22 and IL-23 could play an essential role in recruiting neutrophils to the CF airways and thereby contributing to neutrophil-mediated tissue damage in CF lung disease, referring to CF as a ‘Th17 disease’ (Tan et al., 2011; McAllister et al., 2005; Dubin et al., 2007; Dubin and Kolls, 2011). Combining these results with our data on MDSCs, we propose the following regulatory loop (Fig. 1): (i) P. aeruginosa induces MDSC through a flagellin-mediated mechanism; (ii) MDSCs dampen T cell proliferation and Th17 cell responses and thereby protect P. aeruginosa from T cell-mediated host defense and (iii) MDSCs downregulate neutrophil recruitment by inhibiting IL-17 release. Therefore, MDSCs might limit damage due to exaggerated inflammatory responses. This could be an explanation for our unexpected observation that an increase of MDSCs correlated with improved pulmonary function in chronically P. aeruginosa-infected CF patients (Rieber et al., 2013). The efficacy of MDSC-mediated
An impaired clearance of P. aeruginosa characterizes CF lung disease. P. aeruginosa is recognized through innate pattern recognition receptors, prototypically Toll-like receptors (TLRs), Asialo-GM1 receptors (de Bentzmann et al., 1996) and the NLRC4/IPAF inflammasome (Sutterwala et al., 2007). Among TLRs, P. aeruginosa has been reported to be sensed through TLR2, TLR4, TLR5 and/or TLR9. Recently the role of TLR5 has been highlighted for P. aeruginosa phagocytosis and killing by alveolar macrophages (AMs) (Descamps et al., 2012). Descamps et al. reported, that TLR5, rather than TLR4 engagement is crucial for bacterial clearance by murine AMs in vitro and in vivo. Non-flagellated P. aeruginosa or mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. The clearance of P. aeruginosa by primary murine AMs was linked to increased IL-1 release, which was dependent on TLR5 signaling (Descamps et al., 2012). A few years earlier it was described that TLR5, but not TLR4, receptors are upregulated on human CF airway neutrophils, which was mediated through TLR1/TLR2 signaling (Koller et al., 2008). Another group showed that TLR5 signaling primarily mediates the excessive cytokine production following exposure of human CF airway epithelial cells to P. aeruginosa in vitro (Blohmke et al., 2008). Therefore, targeting TLR5 in CF might be a future therapeutic approach as suggested also by a first population-based genetic modifier study on TLR5 (Blohmke et al., 2010). Bacterial LPS is mainly sensed through TLR4, which has to be under tight regulatory control to prevent hyper-inflammatory responses. After binding LPS, the TLR4/LPS complex is rapidly internalized and targeted for lysosomal degradation in order to restrict inflammatory cascades (Husebye et al., 2006). Particularly, intracellular TLR4 trafficking seems to be dysregulated in CF cells. TLR4 expression and downstream signaling was found to be decreased in human CF airway epithelial cells compared to wild-type airway epithelial cells (John et al., 2010). A subsequent study in CFTR defective murine and human macrophages, however, demonstrated increased TLR4 plasma membrane expression together with abnormal subcellular TLR4 localization. Upon LPS stimulation, CFTR macrophages showed prolonged TLR4 retention in the early endosome and reduced translocation into the lysosomal compartment (Bruscia et al., 2011). The same group showed recently that, in addition, a counterregulatory pathway of LPS signaling involving heme oxygenase-1 is defective in CF macrophages contributing to the exaggerated response to LPS in human in vitro studies and also in vivo in mice (Zhang et al., 2013). Kelly et al. extended these findings of aberrant TLR4 compartmentalization in macrophages to human lung epithelial cells. Contrary to control cells, in CF epithelial cell lines and CF primary nasal epithelial cells TLR4 was not targeted to the lysosome after LPS stimulation, but was persistently expressed on the surface and in the cytoplasm accompanied by a heightened inflammatory response. Summarizing these findings, a dysregulated TLR4 turnover in macrophages might contribute to
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the sustained inflammation, as observed in CF lung disease (Kelly et al., 2013). 3. Immunity in cystic fibrosis: future directions • Functional role of CFTR in myeloid cells: As several myeloid cells have been demonstrated to express CFTR protein (macrophages, neutrophils, and dendritic cells) and CFTR modulators are used nowadays for the treatment of CF patients with G551D mutations (Hoffman and Ramsey, 2013), their effect, particularly of the CFTR potentiator Ivacaftor (Kalydeco), on the homeostasis and functionality of myeloid cells remains to be defined. • Homologies between the porcine and human immune system: The more novel evidence the ferret and porcine CF model reveals, the higher is the interest in the transferability of these data to the human situation. Particularly, the homology of the porcine innate immune response with the human immune system in the context of CF remains to be defined in a disease-specific manner. While studies from the CF pig added several novel insights into the pulmonary pathogenesis, the bacterial clearance deficit and the impaired pH regulation, the in-depth immunological characterization of CF pigs remains elusive, but would be highly relevant for gaining comparative inter-species insights. • Targeting inflammation in CF: Whereas the field of anti-infectives and CFTR modulators moves forward rapidly, drugs dampening inflammation without impairing host defense are rare and the therapeutic development pipeline currently only comprises a few active drugs, namely (i) ibuprofen (with patients), (ii) alpha-1-antitrypsin (protease inhibitor), (iii) Sildenafil (phosphodiesterase inhibitor) and (iv) a small-molecule CXCR2 inhibitor (Moss et al., 2013), see http://www.cff.org/research/ drugdevelopmentpipeline/for updates and more details. Other less established anti-inflammatory approaches include amitryptilin (Nahrlich et al., 2013) and glutathione (Griese et al., 2013). Ibuprofen has been shown to slow the decline of lung function in children with CF, but its broad clinical use is hampered (at least in Europe) by the complex drug monitoring. The general challenge in designing anti-inflammatory compounds for CF is the delicate balance between attenuating overshooting inflammation while preserving the anti-microbial host defense shield. Modulation of resolution, instead of recruitment pathways, may represent a future strategy in CF and other chronic neutrophilic lung diseases. Finally, several questions remain open in the field of innate immunity in CF, particularly regarding specific and safe therapeutic approaches. Consequently, more research is needed to consider innate immune pathways as direct therapeutic targets in CF patients. New animal models and the rapid progress in our understanding of innate immunity beyond CF offers the possibility for young researchers of getting involved in this exciting and rapidly changing area of research. Lessons learned from CF can be extrapolated to other chronic infective lung diseases, such as chronic obstructive pulmonary disease (COPD) or neutrophilic asthma. Vice versa, insights from basic host–pathogen interactions can be applied to the CF-specific context. The way is paved to perform research at the interface between cell biology, immunology, microbiology, lung disease and genetics in order to provide answers to questions that are as complex as CF lung disease is. References Blohmke CJ, Park J, Hirschfeld AF, Victor RE, Schneiderman J, Stefanowicz D, et al. TLR5 as an anti-inflammatory target and modifier gene in cystic fibrosis. Journal of Immunology 2010;185:7731–8.
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Current concepts of immune dysregulation in cystic fibrosis夽 N. Rieber, A. Hector, M. Carevic, D. Hartl ∗ CF Research Group, Department of Pediatrics I, University of Tübingen, Tübingen, Germany
a r t i c l e Article history: Available online xxx Keywords: Cystic fibrosis Immunity Innate Toll-like receptors Neutrophils
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a b s t r a c t Cystic fibrosis (CF) lung disease is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and is characterized by a perpetuated feedback loop of bacterial infection and inflammation. Both intrinsic (CFTR-dependent) and extrinsic (CFTR-independent) mechanisms contribute to the inflammatory phenotype of CF lung disease. Innate immune cells, initially recruited to combat bacterial pathogens, are acting in a dysregulated and non-resolving fashion in CF airways and cause harm to the host by releasing proteases and oxidants. Targeting harmful immune pathways, while preserving protective ones, remains the challenge for the future. This review highlights current concepts of innate immune dysregulation in CF lung disease. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. © 2014 Elsevier Ltd. All rights reserved.
1. Immunity in cystic fibrosis: more questions than answers Cystic fibrosis (CF) lung disease is characterized by a chronic and non-resolving activation of the innate immune system with release of chemokines and an infiltration of neutrophils into the airways. Neutrophil-derived oxidants and proteases cause harm to multiple cellular and humoral factors and have recently been shown to impair Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) functionality itself (Le Gars et al., 2013). Beyond neutrophils, the immune response in CF lungs is dysregulated at several levels, including impaired (i) ceramide homeostasis, (ii) apoptosis, (iii) autophagy, (iv) macrophage polarization and functionality, (v) Tcell responses (Th2/Th17 predominance) and other deviations as reviewed recently (Hartl et al., 2012). Research in innate immunity in CF is aimed at (i) understanding the key mechanisms driving this pro-inflammatory vicious circle, (ii) identifying biomarkers that predict the course of disease and (iii) targeting specific components of the innate immune system as future therapeutics. However, our precise understanding of innate immunity in CF is handicapped by several uncertainties in the field: 1. The chicken-and-egg issue: what comes first? Inflammation or bacterial infection in the early course of CF lung disease? Is there intrinsic CFTR-dependent inflammation also without any prior
夽 This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. ∗ Corresponding author. Tel.: +49 70712981460. E-mail address: [email protected] (D. Hartl).
infection or were our methods to detect infection in previous studies just not sensitive enough to assess microbial impact? Recent insights from culture-independent microbial detection techniques have to be taken into account when addressing this question (Rabin and Surette, 2012). Countries with CF newborn screening and bronchoalveolar lavage (BAL) studies have shed light on this relationship (Sly et al., 2013) and new animal models, primarily the CF pig (Rogers et al., 2008), will help to answer this question. 2. Inter-species differences: Basic immunology relies on murine models to dissect pathways that contribute to disease. However, both (i) the lung anatomy and (ii) the myeloid cell composition differ substantially between mouse and man. New animal models, namely the CF ferret and the CF pig, present major advantages to overcome these hurdles, but studies are required to comprehensively characterize their immune responses and to assess their potential as therapeutically useful model systems to target inflammation in CF lung disease. 3. Who’s bad? Are neutrophils, the most abundant inflammatory cell in CF airways, good or bad? If we deplete them, our first cellular shield against bacteria and fungi is lacking, but there is compelling evidence that neutrophil-derived products, mainly proteases, cause harm to the extracellular pulmonary matrix. Therefore, one might target neutrophil products, for instance proteases. However, these approaches showed limited success so far in clinical studies. As there are several functionally distinct phenotypes of neutrophils (Borregaard, 2010; Kolaczkowska and Kubes, 2013), one could target harmful subtypes while preserving protective ones. But which markers discriminate these subtypes? What is the role of myeloid-derived
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suppressor cells (MDSCs) that dampen overshooting T cell activation (Gabrilovich and Nagaraj, 2009)? What about macrophages? They are required as potent phagocytic cell type, clearing both pathogens and – maybe more important for advanced CF lung disease – apoptotic cells through efferocytosis (Vandivier et al., 2009). Macrophages can be subdivided into classical M1 and M2 macrophages, the latter being induced in a Th2-environment and driving fibrosis and extracellular matrix remodeling. Data on M1/M2 macrophage phenotypes in CF are controversial, so it’s too early to target one of them, at least from our point of view. Data on other innate immune cell types, such as dendritic cells, natural killer (NK) cells, NKT cells and innate lymphoid cells (ILC) are scarce in the field of CF and additional studies are required to define their potential roles and CF disease relevance. 4. Diagnostic consequences: Which biomarkers could indicate patients who are prone to a rapid loss of lung function and should be monitored with greater caution? A recent study shows that free elastase in BAL fluid may represent such a predictive biomarker (Sly et al., 2013). However, BAL is hardly implementable in many clinical CF routines. Elastase in sputum could serve as an alternative, but is not – or hardly – feasible in infants below five years of age and requires standardized pre-analytical processing for reproducible biomarker analyses. 5. Therapeutic consequences: The cycle of inflammatory cell recruitment and unopposed immune cell activation causes tissue damage and leads to irreversible loss of lung function. Proteases and reactive oxygen species (ROS), released by infiltrating myeloid cells, are two key components in this cycle. Consequently, using anti-proteases and/or anti-oxidants are two mechanistic anti-inflammatory approaches in CF lung disease. However, several attempts to target these pathways were only partially successful, presumably due to (i) too low drug concentrations deposited at the pulmonary target site and (ii) inclusion of older CF patients with advanced lung disease characterized by irreversibly-remodeled and – damaged lung structure (Griese et al., 2008, Gaggar et al., 2011). What do we learn from this? How early do we have to start targeting proteases and oxidants to achieve clinical success? What about the compartmentalization of candidate drugs? It is critical that compounds reach their target in the midst of the acidic, oxidative, hyperviscous and hyperproteolytic micro-environment of the CF airway lumen. Are small-molecule compounds superior to natural protease inhibitors or do they show severe side effects as they also inhibit intracellular proteases that are involved in bacterial killing? Accordingly, it would make sense to engineer cell-impermeable compounds. All these issues have to be considered for new drug developments in CF lung disease. In the section below, we summarize and discuss selected recent findings in the field of innate immunity in CF lung disease. 2. Immunity in cystic fibrosis: recent findings 2.1. CFTR in myeloid cells Previous studies provided convincing evidence that human neutrophils express the CFTR protein, localized in the membrane of secretory vesicles (Painter et al., 2006). Optimal microbicidal activity of neutrophils relies on the generation of toxic agents such as hypochlorous acid (HOCl) within phagosomes (Hampton et al., 1998). HOCl formation, in turn, requires chloride ion transported from the cytoplasm into phagosomes mediated by chloride channels (Nauseef, 2007). Studies demonstrated that neutrophils pretreated with a CFTR inhibitor or siRNA-mediated CFTR
knock-down in neutrophil-like HL-60 cells reduced Cl− transport into phagosomes and impaired killing of phagocytosed bacteria (Painter et al., 2006, 2010; Bonvillain et al., 2010). Therefore, CFTR seems to play a role in regulating antimicrobial neutrophil activities. Zhou et al. (2013) recently confirmed and extended these studies by showing that nearly all mature phagosomes of human peripheral blood neutrophils are CFTR positive. The authors further stably expressed enhanced green fluorescent protein (EGFP) together with wt-CFTR or dF508-CFTR, respectively, in a promyelocytic cell line, where EGFP-wt-CFTR associated with phagosomes. In contrast, significantly less dF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Notably, the CFTR corrector compound VRT-325 facilitated the recruitment of dF508-CFTR to phagosomes. When viewed in combination, these data demonstrated the possibility of pharmacologic correction of impaired recruitment of mutant CFTR to the phagosome. This approach might enhance the chloride supply into the phagosomes of neutrophils in CF patients and increase their antimicrobial function (Zhou et al., 2013). However, promyelocytic cell lines, such as HL-60 cells or PLB-985 cells, used in the latter study, are only partially adequate models to study phagocytosis and other neutrophil functionalities, since their vesicle and granule compositions differ from primary human neutrophils and are therefore restricted as cellular model systems when recapitulating the physiological sequence of vesicle and granule membrane fusion events involved in phagocytosis by mature neutrophils. In contrast to patients with primary (inherited) immunodeficiencies in neutrophil function, such as chronic granulomatous disease, CF patients are not affected by recurrent invasive infections in general. Hence, the nature of neutrophil dysfunction in CF patients, has to be more subtle and is probably only of major disease relevance for (i) the extravascular pulmonary site of infection and for (ii) defined stages of host–pathogen interactions, an issue that, however, remains poorly understood. Recent bone marrow transplant studies corroborated the functional impact of myeloid Cftr in vivo (Su et al., 2011). Using conditional inactivation of Cftr in myeloid cells by means of a myeloid-targeted Cre-recombinase, Bonfield et al. demonstrated that the conditional “myeloid CF mice” displayed survival and inflammatory characteristics between wildtype and full genotypic Cftr−/− mice in P. aeruginosa infection models, depending on the stage of infection (Bonfield et al., 2012). In particular, the contribution of myeloid CFTR was most critical at later post-infection time points compared to the whole CFTR-deficient mice, supporting the notion that epithelial CFTR is involved in acute anti-bacterial host defense, while myeloid CFTR comes one step later when myeloid cells have entered the airways. Effects on other CF-associated phenotypes, such as epithelial ion transport, growth reduction or intestinal obstruction, as observed in the total absence of functional Cftr, were not detected in the myeloid-targeted conditional CF mouse model. In contrast to these studies, Oceandy et al. reported earlier in another CF mouse model that Cftr gene complementation in airway epithelium was sufficient to normalize pathogen clearance and inflammatory anomalies (Oceandy et al., 2002), suggesting that epithelial rather than myeloid CFTR is essential. The differing results may be due to the mouse models and the Cftr complementation/targeting methods used. Further comparative studies in mice and men are required to precisely define the contribution of myeloid CFTR to CF lung disease. 2.2. Myeloid-derived suppressor cells Myeloid-derived suppressor cells are innate immune cells generated in cancer and pro-inflammatory microenvironments (Gabrilovich and Nagaraj, 2009). These specialized innate immune
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Th17 suppression may regulate the immunopathogenesis and disease progression in P. aeruginosa infected CF patients. Importantly, P. aeruginosa downregulates its flagellin expression when exposed to airway secretions from CF patients with chronic lung infections (Wolfgang et al., 2004). In addition, flagellin detection by the immune system is also hindered in mucoid strains of P. aeruginosa or within biofilms. Therefore, the effect of P. aeruginosa on MDSCs may depend on several bacterial factors and is probably more complex. Moreover, the exact locations of MDSC–Th17 interactions (blood versus lamina propria versus lumen of the CF lung), the signaling dominance and disease relevance of MDSCs at various stages of CF lung disease remain to be investigated in future studies.
2.3. Toll-like receptors
Fig. 1. Pseudomonas – MDSC – T cell–host-defense circuit in the CF lung. Continuous Pseudomonas – phagocyte contacts result in the release of pro-inflammatory mediators that are capable of driving Th17 generation. Pseudomonas-derived flagellin induces MDSCs that suppress Th17 responses and affect the inflammatory CF circuit at two levels: first, CD4+ T cell proliferation is suppressed resulting in quantitatively less effector T cells. Second, IL-17 release by CD4+ T cells is dampened, leading in combination to a decrease of functional Th17 cells in CF. The pathophysiological consequences of this MDSC–Th17 interaction in the context of host–pathogen interactions and disease progression could be two-fold: on the one hand the reduction of bioactive IL-17 implies a decrease of neutrophil recruitment and phagocytic activity, favoring the survival of P. aeruginosa. On the other hand, MDSCs may dampen excessive Th17-neutrophil activation thereby preventing or limiting auto-destructive host tissue damage mediated through neutrophil-derived proteases.
cells are characterized by their capacity to suppress T-cell responses and thereby modulate adaptive immunity. Consequently, MDSCs are considered as a key intermediary in balancing innate and adaptive immune responses, particularly under chronic disease conditions (Gabrilovich and Nagaraj, 2009). We recently demonstrated that granulocytic MDSCs accumulate in CF patients chronically infected with P. aeruginosa (Rieber et al., 2013). The percentage of MDSCs in the peripheral blood correlated indirectly with CF lung disease activity. Flagellated P. aeruginosa culture supernatants induced the generation of MDSCs, an effect that was mimicked by purified flagellin protein and significantly reduced using flagellin-deficient P. aeruginosa bacteria. Functionally, MDSCs efficiently suppressed polyclonal T cell proliferation and modulated Th17 responses. These studies demonstrated that flagellin induces the generation of MDSCs and suggest that P. aeruginosa utilizes this mechanism to undermine T cell-mediated host defense in CF. Importantly, recent lines of evidence indicate that T helper 17 (Th17) cells and the IL-17 axis-related cytokines IL-22 and IL-23 could play an essential role in recruiting neutrophils to the CF airways and thereby contributing to neutrophil-mediated tissue damage in CF lung disease, referring to CF as a ‘Th17 disease’ (Tan et al., 2011; McAllister et al., 2005; Dubin et al., 2007; Dubin and Kolls, 2011). Combining these results with our data on MDSCs, we propose the following regulatory loop (Fig. 1): (i) P. aeruginosa induces MDSC through a flagellin-mediated mechanism; (ii) MDSCs dampen T cell proliferation and Th17 cell responses and thereby protect P. aeruginosa from T cell-mediated host defense and (iii) MDSCs downregulate neutrophil recruitment by inhibiting IL-17 release. Therefore, MDSCs might limit damage due to exaggerated inflammatory responses. This could be an explanation for our unexpected observation that an increase of MDSCs correlated with improved pulmonary function in chronically P. aeruginosa-infected CF patients (Rieber et al., 2013). The efficacy of MDSC-mediated
An impaired clearance of P. aeruginosa characterizes CF lung disease. P. aeruginosa is recognized through innate pattern recognition receptors, prototypically Toll-like receptors (TLRs), Asialo-GM1 receptors (de Bentzmann et al., 1996) and the NLRC4/IPAF inflammasome (Sutterwala et al., 2007). Among TLRs, P. aeruginosa has been reported to be sensed through TLR2, TLR4, TLR5 and/or TLR9. Recently the role of TLR5 has been highlighted for P. aeruginosa phagocytosis and killing by alveolar macrophages (AMs) (Descamps et al., 2012). Descamps et al. reported, that TLR5, rather than TLR4 engagement is crucial for bacterial clearance by murine AMs in vitro and in vivo. Non-flagellated P. aeruginosa or mutants defective in TLR5 activation were resistant to AM phagocytosis and killing. The clearance of P. aeruginosa by primary murine AMs was linked to increased IL-1 release, which was dependent on TLR5 signaling (Descamps et al., 2012). A few years earlier it was described that TLR5, but not TLR4, receptors are upregulated on human CF airway neutrophils, which was mediated through TLR1/TLR2 signaling (Koller et al., 2008). Another group showed that TLR5 signaling primarily mediates the excessive cytokine production following exposure of human CF airway epithelial cells to P. aeruginosa in vitro (Blohmke et al., 2008). Therefore, targeting TLR5 in CF might be a future therapeutic approach as suggested also by a first population-based genetic modifier study on TLR5 (Blohmke et al., 2010). Bacterial LPS is mainly sensed through TLR4, which has to be under tight regulatory control to prevent hyper-inflammatory responses. After binding LPS, the TLR4/LPS complex is rapidly internalized and targeted for lysosomal degradation in order to restrict inflammatory cascades (Husebye et al., 2006). Particularly, intracellular TLR4 trafficking seems to be dysregulated in CF cells. TLR4 expression and downstream signaling was found to be decreased in human CF airway epithelial cells compared to wild-type airway epithelial cells (John et al., 2010). A subsequent study in CFTR defective murine and human macrophages, however, demonstrated increased TLR4 plasma membrane expression together with abnormal subcellular TLR4 localization. Upon LPS stimulation, CFTR macrophages showed prolonged TLR4 retention in the early endosome and reduced translocation into the lysosomal compartment (Bruscia et al., 2011). The same group showed recently that, in addition, a counterregulatory pathway of LPS signaling involving heme oxygenase-1 is defective in CF macrophages contributing to the exaggerated response to LPS in human in vitro studies and also in vivo in mice (Zhang et al., 2013). Kelly et al. extended these findings of aberrant TLR4 compartmentalization in macrophages to human lung epithelial cells. Contrary to control cells, in CF epithelial cell lines and CF primary nasal epithelial cells TLR4 was not targeted to the lysosome after LPS stimulation, but was persistently expressed on the surface and in the cytoplasm accompanied by a heightened inflammatory response. Summarizing these findings, a dysregulated TLR4 turnover in macrophages might contribute to
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the sustained inflammation, as observed in CF lung disease (Kelly et al., 2013). 3. Immunity in cystic fibrosis: future directions • Functional role of CFTR in myeloid cells: As several myeloid cells have been demonstrated to express CFTR protein (macrophages, neutrophils, and dendritic cells) and CFTR modulators are used nowadays for the treatment of CF patients with G551D mutations (Hoffman and Ramsey, 2013), their effect, particularly of the CFTR potentiator Ivacaftor (Kalydeco), on the homeostasis and functionality of myeloid cells remains to be defined. • Homologies between the porcine and human immune system: The more novel evidence the ferret and porcine CF model reveals, the higher is the interest in the transferability of these data to the human situation. Particularly, the homology of the porcine innate immune response with the human immune system in the context of CF remains to be defined in a disease-specific manner. While studies from the CF pig added several novel insights into the pulmonary pathogenesis, the bacterial clearance deficit and the impaired pH regulation, the in-depth immunological characterization of CF pigs remains elusive, but would be highly relevant for gaining comparative inter-species insights. • Targeting inflammation in CF: Whereas the field of anti-infectives and CFTR modulators moves forward rapidly, drugs dampening inflammation without impairing host defense are rare and the therapeutic development pipeline currently only comprises a few active drugs, namely (i) ibuprofen (with patients), (ii) alpha-1-antitrypsin (protease inhibitor), (iii) Sildenafil (phosphodiesterase inhibitor) and (iv) a small-molecule CXCR2 inhibitor (Moss et al., 2013), see http://www.cff.org/research/ drugdevelopmentpipeline/for updates and more details. Other less established anti-inflammatory approaches include amitryptilin (Nahrlich et al., 2013) and glutathione (Griese et al., 2013). Ibuprofen has been shown to slow the decline of lung function in children with CF, but its broad clinical use is hampered (at least in Europe) by the complex drug monitoring. The general challenge in designing anti-inflammatory compounds for CF is the delicate balance between attenuating overshooting inflammation while preserving the anti-microbial host defense shield. Modulation of resolution, instead of recruitment pathways, may represent a future strategy in CF and other chronic neutrophilic lung diseases. Finally, several questions remain open in the field of innate immunity in CF, particularly regarding specific and safe therapeutic approaches. Consequently, more research is needed to consider innate immune pathways as direct therapeutic targets in CF patients. New animal models and the rapid progress in our understanding of innate immunity beyond CF offers the possibility for young researchers of getting involved in this exciting and rapidly changing area of research. Lessons learned from CF can be extrapolated to other chronic infective lung diseases, such as chronic obstructive pulmonary disease (COPD) or neutrophilic asthma. Vice versa, insights from basic host–pathogen interactions can be applied to the CF-specific context. The way is paved to perform research at the interface between cell biology, immunology, microbiology, lung disease and genetics in order to provide answers to questions that are as complex as CF lung disease is. References Blohmke CJ, Park J, Hirschfeld AF, Victor RE, Schneiderman J, Stefanowicz D, et al. TLR5 as an anti-inflammatory target and modifier gene in cystic fibrosis. Journal of Immunology 2010;185:7731–8.
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