Innate Immune Recognition in Infectious and ... - ATS Journals

State of the Art Innate Immune Recognition in Infectious and Noninfectious Diseases of the Lung Bastian Opitz1, Vincent van Laak1, Julia Eitel1, and Norbert Suttorp1 1

Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charite´–Universita¨tsmedizin Berlin, Berlin, Germany

CONTENTS 1. Introduction 2. Pattern Recognition Receptors as a Basic Part of the Innate Immunity in General 2.1. Toll-like Receptors 2.2. NOD-like Receptors 2.3. RIG-I–like Receptors 2.4. Cytosolic DNA Sensors 2.5. Pattern Recognition Receptors and Control of Adaptive Immunity. 3. Pattern Recognition Receptors in Pulmonary Diseases 3.1. Pattern Recognition Receptors in Innate Immune Responses to Respiratory Tract Infections 3.2. Pattern Recognition Receptors and COPD 3.3. Role of Pattern Recognition Receptors in Sterile Inflammation of the Lung 3.4. Pattern Recognition Receptors in Inflammation and Allergy Affecting the Lung. 4. Conclusions Diseases of the respiratory tract are among the leading causes of death in the world population. Increasing evidence points to a key role of the innate immune system with its pattern recognition receptors (PRRs) in both infectious and noninfectious lung diseases, which include pneumonia, chronic obstructive pulmonary disease, acute lung injury, pneumoconioses, and asthma. PRRs are capable of sensing different microbes as well as endogenous molecules that are released after cell damage. This PRR engagement is the prerequisite for the initiation of immune responses to infections and tissue injuries which can be beneficial or detrimental to the host. PRRs include the Toll-like receptors, NOD-like receptors, RIG-I–like receptors, and cytosolic DNA sensors. The PRRs and their signaling pathways represent promising targets for prophylactic and therapeutic interventions in various lung diseases. Keywords: lung; innate immunity; infection; Toll-like receptor; inflammasome

(Received in original form September 23, 2009; accepted in final form February 17, 2010) Supported by grants given by the Deutsche Forschungsgemeinschaft (OP 86/5-1 and OP 86/7-1), the Ju¨rgen Manchot Stiftung and the Deutsche Gesellschaft fu¨r Pneumologie und Beatmungsmedizin (to B.O.), and by the German Ministry of Education and Research (BMBF) (project B3 in PROGRESS) (to N.S.). Correspondence and requests for reprints should be addressed to Bastian Opitz, M.D., Department of Internal Medicine/Infectious Diseases and Pulmonary Medicine, Charite´–Universita¨tsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: [email protected] Am J Respir Crit Care Med Vol 181. pp 1294–1309, 2010 Originally Published in Press as DOI: 10.1164/rccm.200909-1427SO on February 18, 2010 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Our understanding of mechanisms that promote molecular recognition of microbes and endogenous danger signals by the innate immune system has substantially improved over the last decade. What This Study Adds to the Field

This comprehensive review summarizes the prominent roles of transmembrane and cytosolic pattern-recognition receptors in disease pathogenesis during infectious and noninfectious disorders of the lung.

1. INTRODUCTION The respiratory tract constitutes a large surface of the body in contact with the outside environment. Whereas the pharyngeal mucosa is colonized by microbes that do not necessarily cause strong inflammatory reactions, the lower respiratory tract is considered to be sterile, although some normally nonpathogenic microbes might also be found when using more refined microbiological techniques (1). Invasion, however, of pathogenic microbes into the lower respiratory tract represents a serious threat that requires immediate immune responses. The WHO estimates 429 million cases of acute lower respiratory tract infections in 2004 worldwide, making it the third leading cause of death in the world (2). Moreover, noninfectious and chronic lung diseases also substantially contribute to morbidity and mortality in the world population. Chronic obstructive pulmonary disease (COPD), for example, is the fourth leading cause of death in most industrialized countries (3). The immune system of the respiratory tract with its pattern recognition receptors (PRRs) discussed below plays an indispensable role in both acute and chronic disorders affecting the lung. PRRs include the well-known Toll-like receptors (TLRs), as well as the recently found cytosolic NOD-like receptors (NLRs), RIG-I–like receptors (RLRs), and DNA sensors (4–9). These molecules are expressed in alveolar macrophages, lung epithelial cells, and in intraepithelial dendritic cells (DCs), which come in contact with invading pathogens first, but also in subsequently recruited immune cells. Moreover, PRRs have also been found in endothelial and stromal cells. Different PRRs are generally capable of responding to (1) microbial infections, (2) cell injury–associated endogenous molecules, and (3) large particles such as asbestos fibers (4–7). During infections, the first-line defense in the respiratory tract depends on the barrier function of epithelia, on the tracheobronchial mucociliary system that carries inhaled particles and microbes away from the lower respiratory tract, and on constitutively

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Figure 2. Role of PRRs in the lung. This schematic representation illustrates the key role of different PRRs in the lung homeostasis as well as in infectious and sterile inflammations of the lung.

Figure 1. Overview of the role of pattern recognition receptors (PRRs) in the innate defense to infections in the alveolus. Extra- and intracellular PRRs expressed in alveolar macrophages, epithelial cells, dendritic cells, endothelial cells, and other cell types recognize pathogens. This stimulates production of antimicrobial peptides (AMPs) as well as inflammatory mediators including TNF-a, IL-1b, IL-8, and IFN-b. IL-1b and TNF-a might further activate epithelial cells to produce inflammatory mediators, whereas chemokines such as IL-8 stimulate recruitment of leukocytes. IFN-b activates expression of hundreds of IFN-stimulated genes in an autocrine/paracrine manner which fulfill, for example, antimicrobial functions. PRRs in dendritic cells provide a necessary signal for activating T cell responses.

Furthermore, PRRs are critical regulators of tissue homeostasis and repair in noninflammatory conditions (26–29). A stronger and/or chronic PRR stimulation by microbes, inhaled particles, DAMPs, or possibly even components of tobacco smoke, however, is involved in remodeling and destruction of lung parenchyma, potentially leading to demolition of alveolar walls (emphysema) or interstitial fibrosis (20, 30–32). Cumulating genetic and experimental data additionally indicate that PRRs might be involved in pathogenesis of allergic and granulomatous diseases like asthma and sarcoidosis (33–35).

2. PATTERN RECOGNITION RECEPTORS AS A BASIC PART OF THE INNATE IMMUNITY IN GENERAL 2.1. Toll-like Receptors

expressed antimicrobial peptides, lysozyme, and surfactant proteins (10, 11). In addition, the commensal bacteria in the pharynx may contribute to the defense system by out-competing some pathogenic species, but can also become harmful when aspirated into the lower respiratory tract. The important second- and thirdline defenses are provided by the innate and the adaptive immune responses, both of which, directly or indirectly, depend on the recognition of pathogens by PRRs. PRRs sense microbial infection by recognizing conserved microbial molecules classically defined as pathogen-associated molecular patterns (PAMPs), although nonvirulent microbes do also express some of these molecules. PRR engagement activates the production of inflammatory cytokines, interferons (IFNs), and chemokines on transcriptional and post-translational levels (12), which, for example, activate surrounding cells and regulate recruitment of macrophages and neutrophils (Figure 1). PRRs can regulate cell-autonomous defense mechanisms within, for example, macrophages or epithelial cells that fight intracellular pathogens (13), and the expression of inducible antimicrobial peptides that combat primarily extracellular microbes (14). PRRs on DCs and macrophages further provide an obligatory signal for the induction and shaping of subsequent T cell responses (15–18). These mechanisms explain why PRRs play a key role in acute respiratory tract infections such as pneumonia or infectionassociated exacerbations of chronic obstructive lung diseases (COPD) (11, 19) (Figure 2). Some PRRs respond to large particles such as asbestos fibers or silica crystals and might thus be critically involved in the pathogenesis of pneumoconioses (20, 21). In addition, many PRRs are activated by endogenous, normally intracellular molecules that are released after cell injury. The released endogenous molecules are then called danger-associated molecular patterns (DAMPs). The DAMP recognition by PRRs mediates inflammatory responses to sterile tissue damage and appears to be critically involved in noninfectious inflammations after, for example, lung injuries (22–25).

The 10 members of the human TLR family consist of a cytoplasmic Toll/IL-1 receptor homology (TIR) domain responsible for downstream signaling, and of an extracellular leucine-rich repeat (LRR) domain that most likely mediates ligand binding. TLRs are located at either the cell surface (TLR1, 2, 4–6, 10) or in lysosomal/endosomal membranes (TLR3, TLR7–9) (Figure 3) (4). In the lung, different host cells including macrophages, DCs, lung epithelial cells, and endothelial cells express TLRs. Human alveolar macrophages were shown to express TLR1, -2, -4, -6, -7, and -8, but not TLR3, -5, and -9 (36). Similarly, TLR9 was almost absent from murine alveolar macrophages (37). In contrast, TLR9 as well as TLR7 were highly expressed in plasmacytoid DCs of the human lung. Human myeloid lung DCs are equipped with TLR1–4 (38). Most TLRs, including TLR1–6 as well as TLR9, have also been demonstrated in different tracheal, bronchial, and alveolar epithelial cells (39, 40). Lung endothelial cells express TLR2, -4, -8 and possibly additional TLRs (41, 42), lung fibroblasts have been shown to express TLR2, -3, -4, and -9 (43–46), and airway smooth muscle cells respond to ligands of TLR2, -3, and -4 (47). Different microbial as well as endogenous ligands have been identified for most TLRs except TLR10. TLR2, together with either TLR1 or TLR6, recognizes bacterial tri- or diacetylated lipopeptides, respectively, as well as bacterial lipoteichoic acids, yeast molecules and appears to be involved in the recognition of endogenous hyaluronan and high-mobility group box 1 (HMGB1) (Table 1) (25, 48–55). TLR3 detects double-stranded (ds)RNA, which is an intermediate in viral replication (56), as well as possibly endogenous mRNA released from necrotic cells (57, 58), and TLR7/8 respond to microbial single-stranded (ss)RNA (59, 60). TLR4 is the receptor for lipopolysaccharide (LPS) of gram-negative bacteria as well as for endogenous hyaluronan, HMGB1, oxidized lipoproteins, and oxidized phospholipids (24, 25, 55, 61–64). TLR5 recognizes extracellular bacterial flagellin (65), and TLR9 microbial CpG DNA (66).

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Figure 3. The human Toll-like receptors (TLRs). Overview of the human TLRs as discussed in the text. The TLRs are either located at the cell surface or in endosomal membranes. They respond to infections via sensing of pathogen-associated molecular patterns (PAMPs), and to endogenous molecules (danger-associated molecular patterns [DAMPs]) that are released after tissue damage. The TLRs recruit different adapter molecules and initiate signaling pathways leading to activation of NF-kB–dependent proinflammatory gene expression and/or to IRF3/7-mediated type I IFN (IFNa/b) expression.

The ability of TLRs to activate transcription factors differs and depends on differential engagement of the four TIR domain– containing adapter molecules MyD88 (myeloid differentiation primary response gene 88, which also mediates IL-1 receptor [IL-1R] and IL-18 receptor [IL-18R] signaling), Mal (MyD88adapter-like), TRIF (TIR domain-containing adapter inducing IFN-b), and TRAM (TRIF-related adapter molecule) (4, 67). All TLRs except TLR3 are able to initiate a MyD88-dependent signaling pathway leading to NF-kB–dependent expression of, for example, antimicrobial peptides and proinflammatory mediators such as TNF-a, IL-8, and pro–IL-1b. Whereas TNF-a, IL-8, and other cytokines subsequently regulate the inflammatory response and contribute to leukocyte recruitment, pro–IL-1b needs first to be processed in a caspase-1–dependent second regulatory step (as discussed below). In addition to stimulating NF-kB–dependent gene expression, TLR3, -4, -7, -8, and -9 are capable of activating IRF (IFN regulatory factor) transcription factors and mediating type I IFN responses, which are crucial for such things as the antiviral defense (4, 67). Different TLR genes are highly polymorphic. TLR4 loss-offunction mutations, for example, have been linked with blunted bronchospasm (68) and systemic inflammatory responses (69) to inhaled LPS in human adults. Genetic variations in human TLR2, TLR4, or TLR5 have further been associated with altered susceptibilities to, for example, Mycobacterium tuberculosis, respiratory syncytial virus, or Legionella pneumophila infections, respectively (70–73). In addition, polymorphisms in the key signaling molecules MyD88, IRAK4 (inhibitory kB kinase 4), NEMO (NF-kB essential modulator) and IkBa (inhibitory kB a), which all regulate NF-kB activation downstream of the TLRs and other receptors, affect responses to different infections including pneumococcal and mycobacterial diseases in humans (74–78) (see also below). 2.2. NOD-like Receptors

The NLR family comprises 22 members in humans, and only few of them have been functionally characterized. Most NLRs are located in the cytosol. They all consist of a central nucleotide-binding oligomerization (NOD) domain, and of C-terminal LRRs which possibly mediate ligand binding. In addition, they contain different N-terminal effector binding

domains such as caspase recruitment domains (CARD), pyrin domains (PYD), or baculovirus inhibitor repeats (BIR), and thus activate diverse downstream signaling pathways (5, 79). Among the best-studied NLRs are the CARD-containing molecules NOD1 and NOD2, which both act as cytosolic PRRs. Whereas NOD1 is ubiquitously expressed, NOD2 is mainly expressed in leukocytes but also in lung epithelial cells (80, 81). NOD1 detects bacterial cell wall peptidoglycan containing meso-diaminopimelic acid found primarily in gram-negative bacteria. NOD2 recognizes the muramyl dipeptide (MDP) MurNAc-L-Ala-D-isoGln, which is conserved in peptidoglycans of gram-positive and gram-negative bacteria (82–85). Accordingly, NOD1 contributes to immune responses to different bacteria including Pseudomonas aeruginosa, Chlamydia pneumoniae, Haemophilus influenzae, and L. pneumophila in human as well as murine cells in vitro (86–88) and in mice in vivo (89, 90). NOD2 has been indicated to detect M. tuberculosis, Streptococcus pneumoniae, and C. pneumoniae (80, 89, 91). Both NOD1 and NOD2 activate downstream signaling through the kinase Rip2, leading to an NF-kB–dependent expression of proinflammatory mediators as well as to reactive oxygen species (ROS) production (92–94). With the exception of murine NLRP1, which expresses a CARD, most of the 14 members of the NLRP (NLR family, pyrin domain containing) subgroup of NLRs are characterized by a PYD domain. At least NLRP1–3 form multiprotein complexes called inflammasomes. Inflammasomes, which were first described by Tschopp and colleagues, consist of one or two NLRs, in most cases of the adapter molecule ASC (apoptosisassociated speck-like protein containing a CARD), and of caspase-1 (Figure 4) (12). Inflammasomes respond to various microbial molecules, DAMPs, and inhaled large particles. They regulate a caspase-1–mediated cell death as well as production of the key cytokine IL-1b and of related cytokines including IL18 on a post-translational level. That means that production of IL-1b, in contrast to the release of most other cytokines, is controlled by two signals. The first signal is provided by, for example, the TLRs, which activate an NF-kB–dependent pro–IL-1b expression. The second signal comes from the inflammasomes, which mediate caspase-1–dependent cleavage of pro–IL-1b into mature IL-1b.

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TABLE 1. PRRs,THEIR LIGANDS AND SIGNAL TRANSDUCTION Family TLR

Member

Activated by

TLR1 (1 TLR2) TLR2

NLRP5 NLRP6 NLRP7 NLRP8 NLRP9 NLRP10 NLRP11 NLRP12

Bacterial lipopeptids Bacterial lipopeptids, LTA, oxidized phospholipids, HMGB1 dsRNA, mRNA LPS, oxidized lipoproteins and phospholipids, HMGB1, hyaluronan Flagellin Bacterial lipopeptids ssRNA ssRNA CpG-DNA ? DAP-type PGN MDP ? ? ? Flagellin, bacterial secretion systems Flagellin Lethal toxin, MDP ? E. g. pore-forming toxins, MDP, nucleic acids, ATP, uric acid, hyaluronan ? ? ? ? ? ? ? ?

NLRP13 NLRP14 CIITA RIG-I MDA5 LGP2 ZBP1 AIM2

? ? ? Viral RNA, Pol III-transcribed DNA Viral RNA Viral RNA DNA DNA

TLR3 TLR4

NLR

RLR

DNA sensors

TLR5 TLR6 (1 TLR2) TLR7 TLR8 TLR9 TLR10 NOD1 NOD2 NOD3 NOD4 NLRX1 (NOD5) NLRC4 NAIP5/NAIP NLRP1 NLRP2 NLRP3

Adapter

Activation of

MyD88 MyD88, Mal

NF-kB NF-kB

TRIF MyD88, Mal, TRAM, TRIF

NF-kB, IRF3/7 NF-kB, IRF3/7

MyD88 MyD88 MyD88 MyD88 MyD88 MyD88? Rip2 Rip2 ? ? ? ASC? ASC? ASC ASC ASC

NF-kB NF-kB NF-kB, IRF7 NF-kB, IRF7 NF-kB, IRF7 NF-kB? NF-kB NF-kB ? ? ROS, RLR negative regulation Caspase-1 Caspase-1 Caspase-1 Caspase-1 Caspase-1

? ASC? ? ? ? ? ? ASC?

? Caspase-1? ? ? ? ? ? Caspase-1? NF-kB negative regulation? ? ? MHCII regulation IRF3/7, NF-kB IRF3/7, NF-kB RIG-I, MDA5 inhibition IRF3/7, NF-kB Caspase-1

? ? ? MAVS, STING MAVS — ? ASC

Definition of abbreviations: ASC 5 apoptosis-associated speck-like protein containing a CARD; DAP 5 meso-diaminopimelic acid; dsRNA 5 double-stranded RNA; IRF 5 IFN regulatory factor; MAVS 5 mitochondrial antiviral signaling; MDA 5 melanoma differentiation-associated gene 5; MDP 5 muramyl dipeptide; NLR 5 NOD-like receptor; PGN 5 peptidoglcan; RLR 5 RIG-I–like receptor; ssRNA 5 single-stranded RNA; TLR 5 Toll-like receptor; TRAM 5 TRIF-related adapter molecule; TRIF 5 TIR domain-containing adapter inducing IFN-b.

The first inflammasome to be identified was the NLRP1 (NALP1) inflammasome (12). Human NLRP1 has been detected in different leukocytes as well as in lung epithelium (95). In mice, the NLRP1b gene (one of three genes encoding Nlrp1 in mice) has been linked to sensing of the lethal toxin secreted by Bacillus anthracis (96). The Nlrp1b gene is polymorphic, and only macrophages from mice strains that express functional alleles of Nlrp1b produce IL-1b and undergo cell death after challenge with B. anthracis lethal toxin, while those that express nonfunctional alleles are resistant to the anthrax lethal toxin. Interestingly, the Nlrp1b-mediated macrophage sensitivity to lethal toxin appears to be beneficial in B. anthracis infection in mice in vivo by inducing early proinflammatory cytokine production and neutrophile recruitment (97). Human NLRP1 has also been shown to respond to B. anthracis infection (98), although the exact function of human NLRP1 in B. anthracis infection needs to be further characterized. Further, an NLRP1 inflammasome that also contains NOD2 regulates IL-1b production in response to the peptidoglycan derivative MDP (99). In addition, a recent study investigating expression of inflammasome molecules in monocytes of patients with septic shock found that NLRP1 mRNA levels were linked to survival of patients with septic shock (100).

NLRP3 is expressed in granulocytes, monocytes, macrophages, and DCs (95). The NLRP3 (NALP3) inflammasome mediates a caspase-1–dependent processing of pro–IL-1b as well as pro–IL-18 into their mature forms and regulates a caspase-1–dependent cell death in certain situations. It responds to numerous structurally and chemically diverse stimuli. These NLRP3 activators include microbial RNA and certain forms of DNA, bacterial pore-forming toxins, and MDP (101–105). Accordingly, the NLRP3 inflammasome responds to infections with viruses such as influenza virus, bacteria including Staphylococcus aureus, and fungi like Candida albicans (106–112). Moreover, NLRP3 is activated by necrotic cells, and by uric acid metabolites, ATP, biglycan, and hyaluronan that might be released after tissue damage (102, 113–116). Studies in gene-targeted mice suggest that the inflammatory response to these DAMPs is crucial for the pathogenesis of, for example, acute lung injury and perhaps other lung diseases (23, 117). Finally, the human and mouse NLRP3 inflammasome responds to silica crystals and asbestos as well as to aluminum salts, mechanisms that appear to be critical for the development of pneumoconioses in humans and for the adjuvant effect of aluminum (see also below) (20, 21, 118).

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Figure 4. Role of different inflammasomes in the regulation of IL-1b production. IL-1b production is regulated on a transcriptional and on a posttranslational level. For example, TLR, NOD1/2, and RIG-I–like receptor stimulation can lead to an NF-kB–dependent expression of pro– IL-1b (signal 1). pro–IL-1b needs to be processed into mature IL-1b by the inflammasome complex (signal 2). Inflammasomes consist of caspase-1, ASC in most cases, and of NOD-like receptor (NLR) molecules or AIM2. The NLRs and AIM2 respond to infection via sensing of PAMPs or bacterial virulence factors. In addition, NLRP3 is activated by large particles such as silicia crystals as well as asbestos, and by DAMPs, which might be released by tissue damage related to, for example, acute lung injury.

In addition to the NLRP subgroup of NLRs, NLRC4 (NLR family, CARD domain containing, also called IPAF) as well as NAIP5 (NLR family, apoptosis inhibitory protein 5, also called Birc1e) have also been shown to form inflammasomes. NLRC4 expresses a CARD domain, whereas NAIP5 contains BIR effector domains. Mouse NAIP5 as well as human NAIP have been detected in alveolar macrophages and lung epithelial cells (119, 120). Human NLRC4 is expressed in alveolar macrophages but not in lung epithelial cells (120). The NLRC4 inflammasome recognizes, for example, L. pneumophila and P. aeroginosa flagellin within the host cell cytosol, independently of TLR5 (121–123). NLRC4-deficient mouse cells show impaired IL-1b production after L. pneumophila and P. aeruginosa infection. In addition, NLRC4 cooperates with NAIP5 in mediating a cell-autonomous defense to L. pneumophila (123–128). In mice, different alleles of the Naip5/Birc1e gene determine whether macrophages restrict or support intracellular replication of L. pneumophila, and whether a mouse is resistant or (moderately) susceptible to Legionella infection (125, 128). In resistant mouse strains, a functional NAIP5 mediates recognition of Legionella flagellin, and activation of a caspase1–dependent cell death (pyroptosis), as well as IL-1b secretion (123, 126, 127). Similarly, we recently showed that also the human ortholog NAIP controls intracellular replication of L. pneumophila depending on the recognition of the bacterial flagellin (120). The broadly expressed NLRX1 (NLR family member X1) is the only NLR molecule that is localized in the mitochondrial membrane. NLRX1 mediates production of ROS upon bacterial infection, and negatively regulates RIG-I–like receptor signaling (129, 130). 2.3. RIG-I-like Receptors

The RNA helicases retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) belong to the RIG-I–like receptor (RLRs) family (131, 132). Both proteins show IFN-inducible expression in different host cells, including alveolar macrophages and lung epithelial cells (133, 134). They consist of a DexD/H box RNA helicase domain and two CARDs which mediate signal transduction. MDA5 and RIG-I recognize long dsRNA or shorter dsRNA containing 59-

triphosphate end, respectively, which are specific to viruses and absent from host cells (135, 136). Both helicases signal through the downstream adapter MAVS (mitochondrial antiviral signaling, also called IPS-1, VISA, Cardif) (137–140), which mediates IRF3/7-dependent production of the antiviral type I IFNs as well as NF-kB–dependent induction of inflammatory cytokines. The RIG-I signaling additionally involves STING (stimulator of interferon genes, also called MITA) (141, 142). RIG-I–deficient cells were found to mount greatly diminished IFN-a/b responses to, for example, influenza A virus and respiratory syncytial virus, whereas MDA5 deficiency led to almost abolished cytokine production induced by different picornaviruses (143, 144). The importance of the RLRs for immunity to, for example, influenza A virus infection is underlined by the fact that the virus has evolved mechanisms to counteract RIG-I–induced production of the antivirally acting IFNs in human and murine cells (134, 145, 146). Moreover, RIG-I deficiency also exhibit a severe impact on influenza virus infection in mice in vivo (144). The current concept states that in most cell types, including macrophages, conventional DCs, and (lung) epithelial cells, RIG-I and MDA5 mediate virus recognition, whereas TLRs are crucial for antiviral responses by plasmacytoid DCs (147). In respiratory tract infection in mice, RIG-I in alveolar macrophages and conventional DCs, rather than the TLRs in plasmacytoid DCs, is most important for type I IFN responses to RNA viruses (133). 2.4. Cytosolic DNA Sensors

In addition to viruses, IFN-a/b production can be induced by bacteria that either replicate in the host cell cytosol (148, 149), express secretion systems capable of injecting microbial molecules into the host cell (L. pneumophila, M. tuberculosis) (150–152), or express pore-forming toxins that destruct the phagolysosomal membrane after bacterial phagocytosis (streptococci) (153). It appears that sensing of bacterial DNA within the host cell cytosol is responsible for the type I IFN responses in at least some bacterial infections (152–154). The IFN-b responses induced by the bacteria or by cytosolic DNA stimulation were dependent on IRF3 and possibly STING, but independent of the TLRs and NLRs (141, 150, 152, 153, 155). A recent study indicated that ZBP1 (Z-DNA-binding protein 1,

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also called DAI) serves as a cytosolic DNA receptor capable of inducing IFN-a/b expression in some mouse cells (156). Subsequent studies, however, showed that ZBP1/DAI was not essential for IFN-a/b responses to cytosolic DNA stimulation or infection with intracellular bacteria in most other mouse cell types and human cells (157–159). In addition, we previously showed that human cells infected with L. pneumophila produced IFN-b dependent on MAVS (150). Recent studies elucidated this previous finding by suggesting that Legionella DNA as well as other AT-rich DNA is RNA polymerase III– dependently transcribed into RNA, which is then sensed by RIG-I and MAVS in some cell types (160, 161). Alternatively, another recent study suggested that L. pneumophila RNA, or perhaps an induced host RNA, is recognized by both RIG-I and MDA5 and mediates IFN-a/b responses in mice (162). Overall, polymerase III–RIG-I–MAVS, ZBP1, MDA5, and perhaps additional yet-to-be-identified cytosolic nucleic acid sensors detect microbial DNA and perhaps also RNA in a partly redundant manner in the cytosol of different cell types and activate expression of type I IFNs. Considering that type I IFNs regulate cell-autonomous defense pathways against some intracellular bacteria in vitro (150, 163), as well as immune responses to different bacteria infecting the respiratory tract in mice in vivo (151, 164), an important role of these receptors in bacterial infections of the respiratory tract can be envisioned. In addition, the recently identified cytosolic DNA sensor AIM2 (absent in melanoma 2) does not activate IFN-b expression, but forms a caspase-1–activating inflammasome containing ASC, which regulates IL-1b/IL-18 production (165–168). 2.5. Pattern Recognition Receptors and Control of Adaptive Immunity

PRR signals are involved in discriminating harmless antigens inhaled during respiration from much rarer pathogen-related antigens. PRR activation on antigen-presenting cells promotes up-regulation of costimulatory molecules, and selection of microbial antigens for major histocompatibility complex class II presentation, both necessary for initiating T cell response (169). In addition, different signals from, for example, TLRs, NOD1/2, NLRP3, and the yet-to-be-identified cytosolic DNA receptor(s) might contribute to tailor the T cell response toward Th1, Th2, or Th17 responses (15, 17, 157, 170). Accordingly, NOD2 has been implicated to promote IL-17 production in human memory T cells upon S. pneumoniae infection (18), and TLR2 has been implicated in the Th17 immune response, which cleared nasopharyngeal colonization of S. pneumoniae in mice (171). In influenza A virus respiratory tract infection in mice, RIG-I–MAVS signaling was sufficient to induce a CD81 T cell response, whereas MyD88 was required for the induction of CD41 T cell and antibody responses (172). Moreover, NLRP3 is activated by the common adjuvant alum, which might be required for optimal antibody responses following immunization (118). The knowledge about the role of the PRRs in shaping the adaptive immunity thus helps to develop improved vaccines and clinical trials that evaluate the TLR agonists as adjuvants in, for example, influenza virus vaccination are under way (173).

3. PATTERN RECOGNITION RECEPTORS IN PULMONARY DISEASES 3.1. Pattern Recognition Receptors in Innate Immune Responses to Respiratory Tract Infections

Different PRRs are involved in recognition of extracellular and intracellular gram-positive and gram-negative bacteria as well

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as viruses that cause pneumonia. Children with autosomal recessive deficiencies in MyD88 (the TLR and IL-1/IL-18 receptor adapter) or in the downstream signaling molecules IRAK4 and NEMO suffered from life-threatening infections with S. pneumoniae (77, 78, 174–176), and patients heterozygous for an S180L single nucleotide polymorphism of the TLR2/4 adapter Mal, which impairs its function, are protected against invasive pneumococcal disease (177). MyD88 knockout mice were highly susceptible toward S. pneumoniae infection (178). The phenotypes of single-TLR–knockout mice, however, were less pronounced. Whereas TLR4- and TLR9-deficient mice showed a moderately increased susceptibility toward pneumococcal infections (179–181), TLR2-knockout mice infected with wild-type S. pneumoniae had a modestly reduced inflammatory response in their lungs but an unaltered bacterial load and survival compared with wild-type mice (182). In contrast to infection with wild-type S. pneumoniae, TLR2-deficient mice showed impaired antibacterial defense compared with wild-type mice when infected with S. pneumoniae lacking pneumolysin (183). These studies together indicate an important but redundant role of the TLRs in the immune response to pneumococcal infection. However, the high susceptibility of the MyD88-deficient mice might also be explained by defects in processes initially activated by NLR inflammasomes, which mediate IL-1b/IL-18 production and IL-1/IL-18 receptor activation. This hypothesis should be tested in further studies. In addition to the animal studies in specific knockout mice examining involvement of different PRRs in host defense to S. pneumoniae, other studies indicated a therapeutic potential of synthetic PRR activation. They showed, for example, that treatment of wild-type mice with a TLR2 ligand triggered the innate immune response and improved bacterial clearance and survival in pneumococcal pneumonia (184). Another frequently isolated causative pathogen of pneumonia is the gram-negative, extracellular bacterium Klebsiella pneumoniae. Mice lacking MyD88 showed an impaired inflammatory gene expression and neutrophil recruitment in the lung, a reduced bacterial clearance, and strongly enhanced mortality after infection with K. pneumoniae (185). TRIF-deficient mice demonstrated a similar although less pronounced defect in host defense against Klebsiella. Furthermore, mice deficient in the TLR2/4 adapter Mal were also more susceptible to K. pneumoniae infection (186). Accordingly, TLR4-negative mice showed a reduced proinflammatory gene expression, impaired Th17 responses, enhanced bacterial load in their lungs, and reduced survival after lung infection with Klebsiella (180, 187, 188). Moreover, mice lacking TLR9 demonstrated a reduced survival after intratracheal infection with Klebsiella as well as enhanced bacterial loads in the lungs, the blood, and the spleen (189). Interestingly, the adoptive transfer of bone marrow–derived DC from syngeneic wild-type but not TLR92/2 mice reconstituted antibacterial immunity in TLR92/2 mice, which indicates that TLR9 in DCs is important for defense against this bacterium. In line with a protective role of the TLRs, intratracheal administration of the TLR9 ligand CpG-DNA stimulated a protective immune reaction against K. pneumoniae in mice (190). In addition to the TLRs, NLRP3 contributes to host defense against Klebsiella pneumonia in mice. Mice that lack NLRP3 showed a decreased IL-1b production and inflammatory cell recruitment into the lung (191). Accordingly, these mice as well as mice lacking the inflammasome adapter ASC demonstrated a moderately increased mortality compared with wild-type mice (191). Mice deficient in MyD88 are also highly susceptible to L. pneumophila infection (192–194), whereas mice lacking TLR2 displayed a less severe phenotype (192, 193). Moreover, loss-

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of-function mutation of TLR5 is associated with susceptibility of humans toward Legionnaire’s disease (70), and some studies indicated a role of TLR5 (195) as well as TLR9 (196) in Legionella mouse infection in vivo. Another study, however, did not support these findings (197). Different groups, nevertheless, clearly showed that innate immune responses to L. pneumophila also involved MyD88-dependent but TLRindependent mechanisms including an IL-18R–mediated production of IFN-g by NK cells (192, 194). NAIP5 and NLRC4 mediate growth restriction of L. pneumophila as well as IL-1b/IL-18 production in macrophages of most mouse strains in vitro, and different Naip5 alleles determine whether a mouse is resistant or (moderately) susceptible to Legionella infection in vivo (125, 128). Unlike macrophages of most mice strains, human macrophages support L. pneumophila replication and humans can develop severe pneumonia (Legionnaire’s disease) after Legionella infection. We recently showed that different human cells, nonetheless, possess a related NAIP/NLRC4-dependent cell-autonomous defense mechanism that does not prevent but clearly restricts L. pneumophila replication (120). It is thus an intriguing possibility that other cell-autonomous defense pathways exist, and differ between mice and humans, leading to a permissiveness of human macrophages and to susceptibility of (some) human beings to L. pneumophila pneumonia. Similar to infections with the above-mentioned bacteria, different TLRs are also involved in host defense to P. aeruginosa in a partly redundant manner. Mice lacking TLR2, TLR4, or TLR5 showed no or only a slightly impaired immune response to Pseudomonas infection, whereas a combined deficiency in different TLRs or a knockout in the adapter molecules TRIF and particularly in MyD88 led to strongly impaired immune responses (198–204). Moreover, NLRC4 was required for IL-18 production and early elimination of P. aeruginosa in mice in vivo (205). Like S. pneumoniae, Haemophilus influenzae that express a polysaccharide capsular Haemophilus influenza, that expresses a polysaccharide capsule, colonizes the nasopharynx but is also capable of causing invasive disease. Studies in mice showed that TLR4 and MyD88 are crucial for the host defense against H. influenzae and clearance of this pathogen from the lung (206–208). It was further indicated that nasopharyngeal clearance of encapsulated H. influenzae also required NOD1 signaling in addition to TLR2 and TLR4, whereas individual deficiencies in each of these signaling cascades did not affect clearance of nonencapsulated strains (90). This study is thus an interesting example for the functional crosstalk between the NLR and the TLR pathways. A recent study showed that either the RIG-I–MAVS or the TLR7-MyD88 pathway was sufficient to control initial innate immune responses to intranasal influenza A virus infection. Mice lacking both pathways failed to initiate antivial responses (172). In contrast to the initial innate immunity, the protective adaptive immune response to the virus was governed by the TLR7-MyD88 pathway, but not by the RIG-I–MAVS. In addition, the NLRP3 inflammasome mediates IL-1b/IL-18 production, stimulates neutrophil as well as monocyte infiltration in the lung and controls susceptibility of mice after influenza A virus infection in vivo (106, 109, 111). In contrast to RIG-I, TLR7, and NLRP3, TLR3 appeared to be detrimental in influenza A virus infection in mice in vivo (209). Overall, different extracellular and intracellular bacterial as well as viral pathogens are recognized by multiple PRRs in a partly redundant manner. The cell surface TLRs (e.g., TLR2, -4, and -5) are particularly important for immune responses to extracellular bacteria, but are also involved in host defenses to

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intracellular bacteria. The endosomal, nucleic acid–sensing TLRs (TLR7–9) are key players in viral infections, and appear to contribute to host defenses against bacterial infections. Compared with the TLRs, the role of the cytosolic PRRs in infections is less well understood. NOD1 and NOD2 are key players for host responses to intracellular bacteria like Chlamydia (89) (discussed in Section 2.2), and appear also to contribute to the defense to some extracellular pathogens. The inflammasome-forming NLRs like NLRP3 regulate key cytokines of the immune system (IL-1b, IL-18) as well as necrotic cell death in response to multiple microbial stimuli. They seem to be critically involved in host responses to extracellular and intracellular bacteria as well as viruses, although further in vivo testing is mandatory. The RLRs are key players in viral infections. The role of the RLRs and of the cytosolic DNA sensors in infections with bacteria in vivo remains to be examined. In addition, studies demonstrated that mutations in TLRs and downstream signaling molecules affect susceptibilities of humans toward infectious diseases of the respiratory tract, thus showing that the pathways mainly studied in mice are indeed also critical in humans. These studies should be expanded to genes encoding the cytosolic PRRs and their major signaling molecules. 3.2. Pattern Recognition Receptors and COPD

COPD is a chronic inflammatory disease that leads to irreversible airway obstruction and destruction of the lung parenchyma (emphysema). While cigarette smoking is the primary risk factor for COPD, respiratory infections might also play a role in the development and/or progression of the disease and are the major cause of acute exacerbations (19, 30, 32). Cigarette smoke as well as infections lead to a differential activation of multiple PRRs in lung cells which trigger inflammation and contribute to mucus hypersecretion by epithelial cells, release of proteases by recruited neutrophils, and fibroblast proliferation (3, 210). First, acute exposure to cigarette smoke has been suggested to activate TLR4 in mice and in human cells (31, 211, 212), which might be dependent on a direct recognition of cigarette smoke components, or on epithelial cell injury, release of DAMPs, and recognition of these DAMPs by the PRR. In mice exposed to cigarette smoke, the inflammatory cytokine production and neutrophil recruitment to the lung was dependent on TLR4, MyD88, and IL-1R (31). In addition and/or combined with the above-mentioned mechanism, it is reasonable to speculate that inflammasome activation by DAMPs contributes to pathogenesis of COPD. Inhaled toxic agents, oxidative stress, infections, and necrotic cell death, as well as hypoxia, hypercapnia, focal hypoperfusion, and tissue acidification, might lead to release of DAMPs (e.g., uric acid, ATP) by damaged lung tissue which activates the NLRP3 inflammasome (213). According to this hypothesis, uric acid concentrations were increased in bronchoalveolar fluids of smokers and individuals with COPD (213). Patients with COPD had significantly reduced concentrations of IL-1b antagonists compared with control subjects, and IL-1b concentrations correlated with clinical aspects of disease severity (214). Transgenic mice overexpressing mature IL-1b in the lung epithelium exhibited symptoms that closely recapitulate the features of COPD, including inflammation with neutrophils and macrophages, emphysema, airway fibrosis, and mucus cell metaplasia (215). Moreover, in an experimental model of elastase-induced inflammation and emphysema, IL-1R– as well as MyD88deficient mice, but not different single, double, or triple TLR knockout mice, showed attenuated inflammatory reactions, alveolar wall destruction, and fibrosis (216). The authors of this

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study further demonstrated increased uric acid concentrations in the bronchoalveolar lavage, and found reduced inflammation in mice lacking the inflammasome adapter ASC, or in wild-type mice treated with uricase that degrades uric acid. Neutralization of IL-1 by IL-1Ra (anakinra) substantially attenuated lung inflammation and emphysema induced by elastase, suggesting a therapeutic benefit in chronic lung diseases such as COPD (216). Patients with COPD show increased respiratory tract colonization with, for example, H. influenza, S. pneumoniae, P. aeruginosa, and M. cartarrhalis, which might contribute to chronic inflammation and airway dysfunction. Infection with these and other pathogens including viruses is also a major cause of acute disease exacerbations (19). The increased susceptibility of COPD patients to these infections is poorly understood, but appears to be related to a dysfunctional innate immune system in addition to an impaired mucociliary clearance. According to this hypothesis, alveolar macrophages from smokers and COPD patients were recently shown to express decreased levels of TLR2 (217). In addition, colonizing microbes might also have adapted mechanisms to lower PRRmediated innate immune responses to evade clearance. Moraxella catarrhalis, for example, expresses surface proteins that interact with CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1) resulting in reduced (but not blunted) TLR2-dependent inflammatory responses in human lung epithelial cells, which might enable colonization (218). A continuing low-level innate immune activation, however, might result in damage of the mucosa and parenchyma, thus contributing to COPD pathogenesis. In accordance with a potentially deleterious role of microbial recognition by PRRs in COPD, TLR4 activation with LPS led to emphysema in hamster studies (219). A recent study further showed that cigarette smoke enhanced pulmonary innate immune and remodeling responses to analoga of viral RNA or viral infection in mice (220). Interestingly, these responses were dependent on IL-18 and its receptor IL-18R, a fact that together with the knowledge on regulation of IL-18 by inflammasomes again suggests involvement of these multiprotein complexes in the pathogenesis of COPD. Other studies, however, showed that cigarette smoke might dampen the innate immune responses to microbial TLR3 and TLR4 agonists (221). Moreover, macrophages isolated from bronchoalveolar lavage fluids from smokers produced less TNF-a after exposure to the TLR4 ligand LPS (222), and current or former smoking was associated with significantly reduced levels of antimicrobial peptides in pharyngeal washing fluid and sputum from patients with acute pneumonia (223). Thus, cigarette smoke appears to affect infection-related, PRR-mediated immune reactions with different effects, potentially depending on the infectious agent and PRRs involved as well as other factors. An aberrant and chronically activated innate immune system, however, might contribute to lung remodeling and development of COPD. On the other hand, PRRs also contribute to tissue homeostasis. TLR4-deficient mice were shown to exaggeratedly accumulate reactive oxidants, to exhibit decreased antiprotease activities and cell death in the lung, and to develop spontaneous age-related emphysema (29). TLR4 deficiency in nonhematopoetic cells such as endothelial, epithelial, or fibroblast cells was responsible for emphysema. Treatment with chemical substances that inhibit the generation of reactive oxidant species prevented development of emphysema in mice lacking TLR4 (29). Collectively, different PRR signals play divergent roles in the lung. While a weak constitutive TLR activation appears to be necessary for tissue homeostasis and to avoid emphysema development, TLR, and/or NLR inflammasome activation by cigarette smoke, microbes, and DAMPs contributes to lung

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inflammation and remodeling which might result in COPD. The knowledge of the important role of the PRRs and inflammasomes might help to find new therapeutic approaches by using, for example, substances that reduce DAMP formation (e.g., xanthine oxidase inhibitors) (224) or inhibitors of the inflammasome–IL-1b pathway (IL-1Ra/anakinra, IL-1 Trap/ rilonacept). In addition, the sulfonylurea drug glyburide, which is widely used for type 2 diabetes therapy, has recently been shown to inhibit the NLRP3 inflammasome (225), and thus might also be valuable for the treatment of some inflammatory lung diseases. 3.3. Role of Pattern Recognition Receptors in Sterile Inflammation of the Lung

Long-term exposure to silica or asbestos particles results in occupational lung diseases characterized by pulmonary inflammation and fibrosis as well as in high susceptibility to tuberculosis and risk of developing lung cancer. Recent work showed that silica or asbestos crystals engulfed by resident macrophages activated the NLRP3 inflammasome, leading to IL-1b production (20, 21, 226). Mice deficient in NLRP3 or ASC showed impaired inflammation, granuloma formation, and fibrosis after exposure to silica or asbestos (20, 226). Acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), can arise from a number of insults such as sepsis, gastric acid aspiration, and infections with, for example, highly pathogenic viruses such as H5N1 influenza virus. ALI/ARDS are characterized by a diffuse lung inflammation resulting in noncardiogenic pulmonary edema, impaired gas exchange, and possibly fibrosis, organ failure, and death. Therapy is limited to supportive ventilatory therapy that can further damage the lung and exacerbate the inflammation. In mice models of acid aspiration– and H5N1 influenza virus–induced ALI, TLR4, and TRIF in hematopoetic cells (most likely macrophages) were key for disease development (24). The results of the study suggest that chemical as well as viral agents trigger the oxidative stress machinery, resulting in ROS generation, and local production of oxidized phospolipids, which activate TLR4 and its adapter molecule TRIF. TLR4 and TRIF then stimulate lung inflammation, formation of edema and hyaline membranes, and alveolar wall thickening. Mouse models of hyperoxia-induced ALI also showed that mice deficient in TLR4 (227), TLR2, and TLR4 (25) as well as MyD88 exhibited less inflammation but were more susceptible to lung injury. TLR3-negative mice also exhibited less inflammatory and apoptotic responses as well as less extracellular matrix deposition, but had in contrast to the TLR2-, TLR4-, and MyD88-deficient mice a survival benefit (228). It was speculated that the opposing effects of TLR3 and TLR4 on lung injury were related to an up-regulation of TLR4 in TLR3-deficient mice. Sections from patients with ARDS showed an enhanced TLR3 expression in lung epithelial cells, which does not prove but possibly suggests that TLR3 in lung epithelial cells contributes to the pathogenesis of ARDS (228). Experiments in mice treated with bleomycin to induce lung injury and subsequent fibrosis showed that TLR2/4- or MyD88deficient mice were more susceptible than control mice (25). It was indicated that TLR2 and TLR4 interaction with hyaluronan provided signals that initiate an inflammatory response, protect against epithelial cell apoptosis, maintain epithelial cell integrity, and promote recovery. The authors of this study also showed that circulating hyaluronan fragments purified from human patients with ALI activated mouse macrophages in a TLR2- and TLR4-dependent manner in vitro (25). Other

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studies indicated that bleomycin treatment of mice led to uric acid release by dying cells that triggered NLRP3 inflammasome activation (23, 117). It was suggested that a NLRP3-dependent IL-1b production and subsequent signaling via IL-1R and MyD88 in nonhematopoetic cells were responsible for inflammation, remodeling, and fibrosis. According to this model, bleomycin-induced lung inflammation and fibrosis were dramatically attenuated after treatment of the mice with the IL-1R antagonist anakinra (117) with allopurinol, which impairs uric acid synthesis, and with uricase, which degrades uric acid (23). Overall, all studies conclude that the inflammatory response after lung injury is critically dependent on the release of DAMPs by dying lung cells that activate PRRs. The studies, however, differ in the proposed identities of DAMPs and PRRs involved, and hold partly opposing views in the role of these mechanisms in host protection or pathology. Different lung injury models using acid aspiration, hyperoxia, bleomycin, or other damaging factors in different doses might have led to the release of different DAMPs (e.g., oxidized phospolipids, hyaluronan, RNA, uric acids, or others), which in turn may have activated different PRRs (TLR2, -3, -4, and/or NLRP3). In addition, differences in the time points of the experimental end points (e.g., a few hours after acid aspiration or several days after bleomycin treatment) as well as the backgrounds of the genetically modified mouse strains used might also have led in partly divergent results. However, depending on the magnitude, the PRR-dependent inflammatory responses appear to be involved in both the beneficial maintenance of the structural tissue integrity, repair, and recovery, and in a nonbeneficial development of lung pathology including pulmonary edema formation and/or development of fibrosis. This might be taken into consideration when translating these highly valuable experimental data including the interesting therapeutic approaches in mice (with allopurinol, anakinra) into a clinical perspective. 3.4. Pattern Recognition Receptors in Inflammation and Allergy Affecting the Lung

Sarcoidosis is an inflammatory disorder that most often affects the lung. An analysis of Japanese patients with sarcoidosis found an association between increased diseases susceptibility and a genetic variation in NOD1 (35). Moreover, a recent study found that polymorphisms in NOD2 were associated with severe pulmonary sarcoidosis in white patients (229). Allergic asthma is characterized by airway hyperresponsiveness as well as chronic recurrent airflow obstruction and allergen-triggered airway inflammation. The hygiene hypothesis, raised by David Strachan in 1989, suggests that a general decrease in early exposure to infections, which are sensed by PRRs, skews the balance between Th1 and Th2 immunity toward Th2 responses, promoting the development of type I allergy (230). Accordingly, data obtained from epidemiologic studies suggest that growing up in a farming environment, associated with an abundant exposure toward microbial products, may protect from asthma (231). Moreover, TLR agonists including CpG-DNA have been shown to impair the development of Th2-driven allergic airway disease in some studies (232, 233). Contrary to this hypothesis, however, are experiments in mice which showed that certain respiratory tract infections can promote the pathogenesis of asthma (234). In line with this notion, simultaneous exposure of mice toward LPS and ovalbumin induces allergic sensitization against this allergen (235), and this is mediated at least in part by TLR4- and MyD88mediated activation of airway dendritic cells (235, 236). Fur-

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thermore, antigen exposure during respiratory infection of mice with C. pneumoniae induces airway sensitization via MyD88dependent activation of dendritic cells as well (237). However, PRR-mediated activation of airway epithelial cells may play a central part in allergic airway sensitization as well, since a recent study showed that activation of airway epithelium by a major house dust mite allergen, Derp2, sharing structural homology with the TLR4 cofactor MD2 (238), was required for priming of allergen-specific Th2 responses toward house dust mite extracts (239). So far, data obtained from epidemiologic studies focusing on genetic PRR variants yielded conflicting results, with patients displaying different TLR1, TLR2, TLR4, TLR6, NOD1, NOD2, and NLRP3 variants showing an either increased or reduced risk of developing allergy, allergic asthma, or aspirininduced asthma (33, 34, 240–249). Altogether, oncoming studies investigating gene-targeted allergic mice and SNP patterns of individuals with asthmatic are needed to clarify the functional role of different PRRs in allergic asthma.

4. CONCLUSIONS Different PRRs are able to detect microbial molecules as well as cell injury–associated endogenous molecules, and also to mediate responses to some inhaled large particles. The ensuing immune reactions can be both useful and harmful for the host, possibly depending on their dissemination, magnitude, duration, and ability to eliminate their activators. Accordingly, different PRRs and downstream signaling pathways play key roles in both the regulation of tissue homeostasis and host protection, and in pathology of infectious and noninfectious lung diseases. Moreover, TLR ligands showed therapeutic potential in animal models of allergic airway inflammtion and bacterial lung infections, and improved vaccination efficiencies. Consequently, vaccines against influenza virus and S. pneumoniae that contain TLR agonists as adjuvants, as well as inhalative administration of TLR9 ligands in asthma, are in clinical trials (173, 250) (see also http://clinicaltrials.gov/). Further, the inflammasome–IL-1b pathway has emerged as a promising therapeutic target. The novel understanding of the important role of the NLR inflammasomes and of the inflammasome-regulated cytokine IL-1b in inflammation and disease has already led to novel therapeutic approaches of lung-unrelated chronic diseases including gouty arthritis and type 2 diabetes using the IL-1R antagonist anakinra (251–253). Considering that inhibition/modulation of the inflammasome–IL-1b pathway with anakinra, allopurinol, or rilonacept led to reduced inflammation and pathology in mouse models of acute lung injury and chronic lung diseases, it is an intriguing possibility that these approaches might also show therapeutic benefits in human patients with lung diseases. Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgment: The authors thank Nicolas Schro¨der, Guido Heine, and members of the lab for helpful discussions and valuable suggestions.

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