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Bacterial lung diseases are a major cause of morbidity and mortality both in immunocompromised and in immunocompetent individuals. Neutrophil accumulation, a pathological hallmark of bacterial diseases, is critical to host defense, but may also cause acute lung injury/acute respiratory distress syndrome. Toll-like receptors, nucleotide-binding oligomerization domain (NOD)-like receptors, transcription factors, cytokines, and chemokines play essential roles in neutrophil sequestration in the lungs. This review highlights our current understanding of the role of these molecules in the lungs during bacterial infection and their therapeutic potential. We also discuss emerging data on cholesterol and ethanol as environmentally modifiable factors that may impact neutrophil-mediated pulmonary innate host defense. Understanding the precise molecular mechanisms leading to neutrophil influx in the lungs during bacterial infection is critical for the development of more effective therapeutic and prophylactic strategies to control the excessive host response to infection.
This review highlights the mechanisms underlying neutrophil influx in the lungs and discusses the potential avenues to attenuate neutrophil-mediated lung damage during bacterial pneumonia.
The respiratory system continually encounters microorganisms. The pathogenicity of a microorganism not only depends on its virulence factors, but also on the host's immune defense and the environment. The myeloid and structural/resident cells of the respiratory system detect invading microorganisms by binding their pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs) to bacterial ligands. This recognition triggers a cascade of events leading to the activation of transcription factors, production of chemokines/cytokines, up-regulation of cell adhesion molecules, phagocytic cellular infiltration, and subsequent clearance of the microorganisms. The major players of the innate immune system are neutrophils, macrophages, and dendritic cells. Dendritic cells and macrophages not only produce proinflammatory mediators but also present antigens to induce eventual adaptive immune response. The innate immune system in vertebrates confers immediate defense against invading microbes via mechanisms such as phagocytosis, whereas the adaptive immune system armed with T and B lymphocytes plays an important role in chronic or recurrent infections.
Pneumonia is an important cause of mortality both in developed and in developing countries (1). When microbial infections overwhelm the innate immune defense, the result is pneumonia, which is associated with extensive lung pathology (1, 2). Although pneumonia can be caused by a variety of microbes, such as bacteria, viruses, and fungi, we have primarily focused upon bacterial pneumonia in this review. The initial phase of bacterial pneumonia is characterized by neutrophil-mediated inflammation (1, 2). While neutrophilic inflammation aids in removal of bacteria, it also induces bystander injury to the lung. When severe, this injury may lead to a clinical condition termed acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) (1, 2). It is therefore imperative to explore the molecular mechanisms that underlie pneumonia and ALI/ARDS to formulate therapeutic strategies to augment host immune mechanisms to combat microorganisms while attenuating excessive parenchymal damage.
This review addresses our current understanding of the innate immune responses of the host against different gram-positive and gram-negative bacteria during the acute phase of bacterial pneumonia, with special emphasis on the role of PRRs such as TLRs and NLRs, transcription factors, cytokines, and chemokines in neutrophil accumulation. Furthermore, we discuss emerging roles for cholesterol and alcohol in neutrophil recruitment to the lung during bacterial pneumonia.
A large pool of neutrophils is stored in the bone marrow. Granulocyte-colony stimulating factor (G-CSF) stimulates the proliferation and differentiation of the granulocytic lineage in the bone marrow into mature neutrophils. These neutrophils are mobilized to the bloodstream during bacterial infections and are believed to be the first line defenders of the innate immune system against microbes. To reach sites of inflammation in various organs, including the lungs (3), neutrophils in the bloodstream adhere to the vascular endothelium and migrate across the endothelium. This process involves multiple adhesion molecules from different families that are expressed on the surface of endothelial cells in response to different cytokines/chemokines. As these cells migrate to inflammatory foci, they get activated, generate free radicals, release granule contents, and phagocytose and degrade microbes. Neutrophils are the first phagocytic cells recruited to a site of bacterial infection, but they have a very short life span (<6 h) after release from the bone marrow.
Neutrophils are one of the critical contributors to host defense in the lungs, since (1) selective depletion of neutrophils results in substantial reduction in the clearance of Streptococcus pneumoniae, Klebsiella pneumoniae, and Legionella pneumophila; and (2) repletion of neutrophils in neutropenic mice improves host defense and survival in response to bacterial infection (4–6). Although the neutrophil is the critical cell type implicated in the pathogenesis of ALI/ARDS, its short life span renders it difficult to investigate the signaling cascades in response to stimuli using genetic manipulations. In this context, a novel method has recently been developed to generate mature neutrophils from bone marrow–derived progenitor cells via long-term bone marrow culture system (7). These neutrophils have been shown to be mature as determined by morphology, expression of surface markers (Gr1, CD11b, CD62L, and CXCR2), and functional capabilities, including superoxide generation, exocytosis of granular contents, chemotaxis, phagocytosis, and bacterial killing. Furthermore, these in vitro mature neutrophils are capable of migrating to inflammatory sites in vivo. We anticipate that this system will serve as an important tool to advance our knowledge in neutrophil biology in the future.
Despite the fact that various animal model systems are used to delineate molecular mechanisms underlying neutrophil accumulation and lung injury, murine models have been used extensively because (1) bacteria can induce substantial neutrophil influx and subsequent damage to the lungs of mice during pneumonia, (2) several mouse strains with a variety of gene deletions or disruptions are available, and (3) there is ample availability of reagents and pharmacologic agents to dissect out the signaling mechanisms in murine models.
The innate immune system is responsible for recognizing invading pathogens and subsequent initiation of the inflammatory response and/or host defense. Distinguishing self from non-self is therefore an important hallmark of the immune system. The recognition of microbes relies on PRRs that can recognize molecular moieties common to microbes. The discoveries of both membrane-bound and cytoplasmic PRRs, including TLRs, NLRs, mannose receptors, and RNA helicases have stimulated investigations on the biology of the innate immune system. We have highlighted the roles of TLRs and NLRs in this review, since these receptors have recently been identified and have important roles in host defense against bacterial infections.
A single microbe generally has a variety of molecules called pathogen-associated molecular patterns (PAMPs) that can activate a single or multiple PRRs (8). The intracellular signaling cascades initiated by these receptors may lead to the activation of transcription factors, such as nuclear factor (NF)-κB, activating protein (AP)-1, signal transduction and transcription (STAT) proteins, and interferon regulatory factor (IRF) families (8), thereby regulating the expression of proinflammatory mediators, such as cytokines/chemokines, and adhesion molecules. In turn, these proinflammatory mediators can induce infiltration of neutrophils via enhancing chemotaxis and activating neutrophils to release more cytokines/chemokines. While neutrophils combat pathogens in a nonspecific manner as innate immune cells, antigen-presenting cells, such as dendritic cells and macrophages, present antigens to T lymphocytes to induce antigen-specific adaptive immune response. However, recent reports have suggested that neutrophil-derived IL-18 together with dendritic cell–derived IL-12 can induce IFN-γ synthesis in NK cells in response to intravenous L. pneumophila infection in mice (9). These recent findings reveal a new role for neutrophils in the control of bacterial infection, in addition to their classical phagocytosis/microbicidal functions.
TLRs are type 1 transmembrane receptors, with an ectodomain composed of leucine-rich repeats (LRRs), and are members of a larger superfamily of interleukin-1 receptors (IL-1Rs). Toll was initially identified in Drosophila as a receptor essential for dorsoventral polarity during embryogenesis, and later was shown to also be important for antifungal host defense in insects. The members of this family share a conserved region of approximately 200 amino acids in the cytoplasmic region known as the Toll/IL-1R (TIR) domain (10), whereas the extracellular LRR region is diverse in nature and is directly involved in the recognition of microbes.
TLRs function as dimers, usually forming homodimers except for TLR2, which dimerizes with either TLR1 or TLR6, giving rise to different ligand specificity. To date, 12 TLRs in mice and 10 in humans have been identified (8). Several TLRs can recognize bacteria and/or their components. For example, TLR1, TLR2, and TLR6 recognize lipid and carbohydrate compounds, including lipoteichoic acid and lipoprotieins, from gram-positive bacteria. TLR4 recognizes lipopolysaccharide (LPS), a cell-wall component of gram-negative bacteria and MD-2 is a key molecule important for the TLR4 recognition of LPS, whereas TLR5 recognizes bacterial flagellin. TLR3, -7, -8, and -9 are receptors for nucleic acid and its derivatives. Furthermore, TLR11 has shown to be involved in the recognition of profilin and uropathogenic bacteria (Figure 1).
Several studies have unequivocally demonstrated the role of TLRs in pulmonary host defense (Table 1 and Figure 1). For instance, TLR2 has been shown to be essential for host defense against Streptococcus pneumoniae (11) and Porphyromonas gingivalis (12); however, TLR2 only mediates partial resistance to L. pneumophila (13–15). TLR4 contributes to a protective immune response against both S. pneumoniae (16) and K. pneumoniae (17), although its role is much more pronounced against K. pneumoniae. TLR4 also contributes to pulmonary host defense against Haemophilus influenzae (18). Also, MD-2 plays an important role during Escherichia coli–induced pneumonia (19). Both TLR2 and TLR4 have been shown to be significant for host defense against Acinetobacter baumannii (20) and Pseudomonas aeruginosa (21). TLR5 is an important regulator of neutrophil infiltration into the lung at early time-points (6 h), but not at late time points (24 h or beyond) during L. pneumophila infection (22). TLR9 is required for effective host defense not only against gram-negative pathogens, such as L. pneumophila (23) and K. pneumoniae (24), but also gram-positive pathogens, such as S. pneumoniae (25). These observations reveal (1) the activation of multiple TLRs in response to bacterial infection and (2) the time-dependent activation of TLRs in response to bacterial interaction.
LRRs of TLR2,-4,-5, and -6 are located outside of the cell, while the Toll–interleukin (IL)-1 receptor homology (TIR) domain is located inside the cell in all TLRs (10). Distinct adaptor molecules, including MyD88, TIRAP, TRAM, TRIF, and SARM, physically associate with the TIR domain of TLRs to transduce the signals. MyD88 is important for TLR1, -2, -4, -5, -6, -7, -8, -9, -10, and -11 mediated signaling network, and it is recruited to the TLR complex by TIRAP in TLR2 and TLR4 initiated cascades. TRIF is involved in TLR3 and MyD88-independent TLR4 signaling. TRAM plays a key role in TRIF-dependent, MyD88-independent signaling through TLR4 (Figure 1).
It is important to note that individual TLRs can activate overlapping as well as distinct signaling cascades, ultimately providing diverse biological responses via activation of mitogen-associated protein kinases (MAPKs), and transcription factors, in turn resulting in the expression of growth factors, cytokines/chemokines and cell adhesion molecules (Figure 1). For example, MyD88-dependent TLR signaling cascades lead to early NF-κB activation, whereas MyD88-independent TLR signaling pathways result in delayed NF-κB activation. Of the TLR signaling cascades, the role of interleukin-1 receptor–associated kinases (IRAKs) in host defense has been well established. Four different IRAKs (IRAK-1, IRAK-2, IRAK-M, and IRAK-4) have been identified in mice and humans. Recently, patients with inherited IRAK-4 deficiency were reported who failed to respond to IL-1, IL-18, or to stimulation with TLR2, TLR3, TLR4, TLR5, and TLR9 agonists (26). In addition, findings with IRAK-M gene–deficient mice have shown that IRAK-M serves as a negative regulator of IL-1R/TLR signaling, and therefore, IRAK-M knockout mice showed more neutrophils and augmented bacterial clearance to P. aeruginosa in the lungs in a sepsis model (27).
As most of the TLR studies have been performed in murine models, the efficacy and safety of TLR therapies may not extrapolate to human responses. This is because of (1) differences between the human and murine immune system; (2) differences in the activation profile of human and mouse, such as TLR8 (8); and also because (3) murine investigations are performed on inbred strains that have minimal genetic variation. Though TLR9 agonists, such as CpG oligodeoxynucleotides, have been shown to protect against numerous infectious agents in murine models (28), no human clinical studies have been reported, to our knowledge, using TLR9 agonists in bacterial infections. Since TLR3, TLR7, TLR8, and TLR9 can be activated upon intracellular bacterial infection, resulting in the production of the IFN-α (29), these receptors can be targeted to control bacterial infections.
Investigations have shown that TLR adaptor proteins play an important role in host defense against bacterial pathogens in the lung (Table 1). For instance, MyD88 is critical for host defense against numerous pathogens, including S. pneumoniae (25), H. influenzae (30), P. aeruginosa (31–33), Staphylococcus aureus (32), K. pneumoniae (31), L. pneumophila (14, 33, 34), and E. coli (31). Furthermore, TIRAP is reported to be essential for host defense against E. coli and K. pneumoniae (6, 35). In addition, MyD88 gene–deficient mice show more pronounced phenotype as compared with single or double TLR gene–deficient mice (14, 21, 34). Moreover, MyD88-independent, TRIF signaling has also been shown to be important host defense in the lungs against E. coli (35) and P. aeruginosa (36). These observations demonstrate (1) the importance of MyD88 as an adaptor molecule for several TLRs, (2) the importance of MyD88 and TRIF as adaptor molecules for TLRs, and/or (3) the sequential activation of several TLRs during bacterial infection in the lungs. Our studies using TRIF-blocking peptide to attenuate the expression of IL-8, IL-6, and TNF-α in response to E. coli demonstrate the importance of TRIF in humans (35). These results reveal the potential for using cell-permeable compounds to attenuate cytokine/chemokine production and thereby possibly be useful for reducing excessive neutrophil recruitment to the lungs.
NLRs are the other types of PRRs involved in the innate immune system and are responsible for detecting pathogens and/or PAMPs. NLRs regulate both inflammation and apoptosis. To date, 22 NLR family members in humans have been reported (37). They have been further classified into subfamilies, including NODs and NALPs, the MHC Class II transactivator (CIITA), IPAF, and BIRC1. NLRs are characterized by the presence of a central NOD domain, a C-terminal LRR, and an N-terminal domain, including CARD or the putative protein–protein interaction (PYRIN) domain (37). The LRRs of NLRs are important for pattern recognition of microbial ligands.
Of the NODs, NOD1, and NOD2 are known to recognize bacteria and/or their components. NOD1 and NOD2 recognize the bacterial peptidoglycan components γ-D-glutamyl-mesodiaminopimelic acid and muramyl dipeptide, respectively (37). The NODs interact with microbial molecules by means of a C-terminal LRR region and activate downstream gene transduction events through N-terminal CARD domains, which leads to apoptosis. These receptors transmit the signals by the serine threonine kinase CARDIAK/RICK/RIP2 and activate NF-κB. Microbial molecules are detected by these proteins, resulting in their oligomerization and activation of caspases (37). Active caspases play an important role in inflammation via cleavage and maturation of proinflammatory cytokines. Recent investigations have shown that NOD1 is important for bacteria-induced host response. For example, NOD1 detects P. aeruginosa peptidoglycan, leading to NF-κB activation. Cytokine secretion kinetics and bacterial killing are altered in NOD1-deficient cells infected with P. aeruginosa in the early stages of infection (38). When NOD1 and NOD2 gene–deficient mice were infected intravenously with Listeria monocytogenes, they showed reduced NF-κB activation, and attenuated cytokines/chemokines and neutrophil influx in the liver and spleen (39). They also observed increased bacterial burden in the liver and spleen of NOD1 and NOD2 gene–deficient mice.
NALPs are the other members of the NLR family. NALPs have a PYRIN domain instead of a CARD domain at the N-terminus. This subfamily consists of 14 proteins, but most of their functions have not yet been determined. NALP1, NALP2, and NALP3 have been involved in the cleavage of pro–IL-1β and pro–IL-18 to mature forms via caspase-1 (40). These NALPs form a complex of proteins called an “inflammasome,” which comprises adaptor proteins and caspases. NALPs have emerged as mediators of antibacterial host defense in recent years. NALP1 has been shown to be important for host defense against Bacillus anthracis (40). NALP3 is well studied and has been shown to be essential for host defense against L. pneumophila (41) and Francisella tularensis (42). IPAF is another NLR family member. It contains CARD domains and has been shown to recognize Salmonella flagellin and induce IL-1β production (43). Flagellin of L. pneumophila is also responsible for IPAF-dependent caspase-1 activation (44). NALP5/NAIP5 (Birc1e) is an NLR protein that regulates host susceptibility to the intracellular pathogen L. pneumophila. NALP5 has been implicated in L. pneumophila–induced caspase-1 activation (44). Further understanding of the molecular pathways of NLRs in lung may lead to new strategies for controlling bacterial infectious diseases in the lungs.
Transcription factors are sequence-specific DNA-binding proteins that bind to the promoter or enhancer regions of specific genes and control the transfer of information from DNA to RNA. Although several transcription factors, including NF-κB, AP-1, STAT, and IRFs, play important roles in inflammation, NF-κB and STATs have been demonstrated to play important roles in bacterial pneumonia. NF-κB, a protein complex found in almost all mammalian cell types, is the most studied transcription factor to date. NF-κB exists in homodimeric and/or heterodimeric forms and consists of five family members, which include p50 (NF-κB1), p52 (NF-κB2), c-Rel, RelB, and RelA (p65) (45). All members of the family share the Rel homology domain. NF-κB acts as a transcription factor in response to stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. In normal cells, NF-κB dimers are present in the cytoplasm and inhibited by IκBs (Inhibitor of κB), proteins that contain multiple copies of a sequence called an ankyrin repeat. The IκB proteins mask the nuclear localization signals of NF-κB proteins and keep them in an inactive state in the cytoplasm. Degradation of IκB leads to nuclear translocation of NF-κB and its subsequent binding to the promoters of and “turn on” expression of specific genes.
NF-κB can be activated by TLRs, NODs, and NALPs, and this triggers the expression of proinflammatory genes, including different cytokines and chemokines through MyD88-dependent and -independent cascades. Recently, it was found that NLRs also activate NF-κB. Activation of NF-κB contributes to neutrophil accumulation elicited in the lungs by LPS, which is largely in part through the up-regulation of several neutrophil chemokines (keratinocyte-derived chemokine [KC], macrophage inflammatory protein [MIP]-2, and CXCL5). Studies demonstrate that endogenous NF-κB protects the mice during E. coli (46, 47) and pneumococcal pneumonia, and is essential for the survival during these infections (48). Neutrophil emigration to the alveoli during LPS-induced inflammation was severely reduced in TNF Receptor 1/RelA-double knockout mice, when compared with their control (TNFR1-deficient) mice (49). In a similar manner, it has been shown that deficiency of TNF-α and IL-1 receptor signaling reduces NF-κB activation to S. pneumoniae (49). Furthermore, TRIF-deficient mice also showed reduced NF-κB activation and chemokine expression in the lungs during P. aeruginosa infection (36).
Although the role of NF-κB has been studied extensively in pneumonia, there are other transcription factors, such as STATs, that also play an important role in the innate immunity. STATS can be activated by cytokines, including IL-6. Cytokines bind to their homodimeric or heterodimeric receptors, which can bind to janus kinases (JAK). JAKs can be activated by transphosphorylation followed by phosphorylation of cytokines receptors, allowing STATs to bind cytokine receptors. The activated STATs translocate to the nucleus, where they bind DNA and induce transcription of genes. Studies have shown that the STAT4 is an integral component of the host innate immune response leading to cytokine production and bacterial clearance during K. pneumoniae infection in the lung (50). STAT4 also contributes to P. aeruginosa–induced inflammation, but is not essential for bacterial clearance (51). During E. coli pneumonia, IL-6 family members specifically activate alveolar epithelial STAT3 to promote neutrophil infiltration into the lungs (52).
Cytokines are polypeptides produced by variety of cell types. They have autocrine, paracrine, or endocrine functions to regulate inflammation and immune defense. Cytokines bind to their cognate receptor and signal via second messengers, thereby increasing or decreasing the expression of other membrane proteins, and can be categorized as proinflammatory or anti-inflammatory. TNF-α, IL-1β, IL-6, IL-8, and IFN-γ are major proinflammatory cytokines that participate in acute inflammation; IL-8 acts as a chemoattractant, whereas TNF-α and IL-1β stimulate antigen presentation and increase the expression of cell adhesion molecules. On the other hand, IL-10, TGF-β, and IL-1Ra are major anti-inflammatory cytokines that down-regulate the inflammatory response in the lungs (53).
The importance of IL-12 in antibacterial host defense has been widely established. The IL-12–IFN-γ axis is known to be involved in host immune defense against bacterial pathogens and induces cell-mediated immune responses to clear infection. However, the role of the IL-23–IL-17 axis has only recently been recognized, as mice that lack both IL-12 and IL-23 are more susceptible to bacterial infections (54). IL-23 is a newly described cytokine synthesized by macrophages and dendritic cells. It shares the p40 subunit with IL-12, an important cytokine in the induction of the Th1 response (e.g., IFN- γ), but has a distinct p19 subunit. In general, IL-23 produces more IL-17 from CD4+ T cells than IL-12. Several recent studies suggest that bacterial infection can induce IL-23 expression by macrophages and/or dendritic cells. It has been documented that IL-23 is essential to mediate host defense in the lungs against K. pneumoniae and P. aeruginosa (55, 56).
IL-17, which is mainly produced by T cells via the Th17 immune cascade, has been proposed as a proinflammatory cytokine (57). Recently, five new IL-17 family members have been identified: IL-17B, IL-17C, IL-17D, IL-17E (also named IL-25), and IL-17F (57). Virtually all cells bear the IL-17 receptor, which has been shown to recruit neutrophils to the lung by stimulating the production of the ELR+CXC chemokines, IL-8 or KC (a mouse homolog of IL-8) (57), and G-CSF. In addition, IL-17A seems to be important for the early and the late phase of neutrophil accumulation in the lungs (52). Furthermore, IL-17 can induce the up-regulation of other cytokines, including IL-1β and IL-6, and ICAM-1 by several types of cells (e.g., endothelial cells, epithelial cells) (57). Using gene-deficient mice, it has been shown that IL-17 is important for host defense in the lungs against respiratory infection in mice caused by K. pneumoniae (58).
Cytokines that act as chemoattractants for other cells are known as chemokines. Chemokines are produced locally at sites of infection/inflammation and regulate the recruitment of specific subpopulations of leukocytes from the bloodstream into tissues. Chemokines enhance neutrophil adhesion and extravasation across the postcapillary venules and direct migration neutrophils to sites of infection/inflammation. According to the composition of a cysteine motif located near the N-terminus of these molecules, they are categorized into the C, CC, CXC, and CX3C subgroups. These chemokines have four cysteine molecules, the first two of which are separated by a nonconserved amino acid, and can be further categorized by the presence of the ELR (glutamic acid-leucine-arginine) motif immediately preceding the CXC sequence (59). All known ELR+CXC chemokines are neutrophil chemoattractants. Seven chemokines (IL-8; NAP-2; GRO α, β, and γ; ENA-78; and GCP-2) have been identified in humans (59). Among these, IL-8 is the most potent neutrophil chemoattractant in bacterial pneumonia. Although a real homolog of human IL-8 has not been identified in rodents, KC, MIP-2, LPS-Induced CXC Chemokine (LIX; CXCL5), and lungkine are important chemoattractants in mice in response to bacterial lung infection (60).
While most chemokines are secreted primarily by myeloid cells, lungkine and LIX are secreted by bronchial epithelial cells and type II alveolar epithelial cells, respectively. There are two receptors identified for CXC chemokines: CXCR1 and CXCR2, which are expressed in both humans and mice. CXCR2 binds to all ELR+CXC chemokines and is essential for neutrophil infiltration into the lungs during bacterial infection. In this context, in vivo studies have shown that neutrophil recruitment induced by P. aeruginosa (61), Norcardia asteroids (62) and L. pneumophila (63) involves the CXCR2 receptor (Table 1). Regarding the CXCR2 ligands, KC and MIP-2 are important for neutrophil influx during infection, as blocking these ligands impairs host defense (61, 64). Although lungkine is not important for neutrophil influx into the lung parenchyma, it is important for neutrophil accumulation in the airspaces (65) (Table 1). It has been reported that CXCL5 up-regulation increases neutrophil trafficking during infections with P. aeruginosa, K. pneumoniae, L. pneumophila, and Bordetella bronchiseptica. Subsequent studies have shown that LIX is an important molecule for neutrophil influx in the lungs during LPS-induced inflammation (66). However, the role of LIX in neutrophil infiltration in the lungs during bacterial infections remains to be determined.
Cholesterol exerts complex effects upon the proinflammatory functions of macrophages, PMNs, and other cell types. Cholesterol overloading induces macrophages to produce cytokines, in part through endoplasmic reticulum stress (67–69), though it may also either enhance or attenuate macrophage responses to LPS (70, 71). Hypercholesterolemia primes PMNs for oxidant and granule protein release (72, 73), induces PMN adhesion to and emigration from postcapillary venules (74), and promotes mononuclear cell accumulation in vascular lesions by inducing endothelial chemokines (75, 76). The net effect of systemic dyslipidemia on organ-localized inflammation is likely, however, to be complex, as cholesterol loading also impairs leukocyte chemotaxis (77), and lipoproteins, the particles that carry cholesterol in the bloodstream, bind to and neutralize bacterial LPS (78) and inhibit leukocyte signaling responses to multiple TLR ligands (79). Further complicating expectations for the net effect of cholesterol upon inflammation in the lung in particular is the fact that the mechanism of, and requirements for, PMN transmigration from the vascular compartment into the airspace differ substantially from those at work in other organs (3).
While little is known about either the sensitivity of lung-resident cells to systemic dyslipidemia (i.e., circulating lipoprotein cholesterol) or the lung's local regulatory mechanisms for cholesterol, emerging reports interestingly do suggest that cholesterol trafficking and inflammation may be coupled uniquely in the lung. For example, genetic deletion of the cellular cholesterol efflux pump ATP-binding cassette transporter G1 (ABCG1) induces recruitment of multiple leukocyte subtypes, including PMNs, to the unexposed murine lung (67, 69), responses that reflect cooperative proinflammatory contributions from cholesterol-overloaded alveolar epithelium and alveolar macrophages. Conversely, the potential for therapies that reduce cellular and serum cholesterol to reduce PMN recruitment to the airspace has also been reported. Three different hydroxy-methylglutaryl coenzyme A reductase inhibitors (“statins”) used to treat clinical hypercholesterolemia have now been shown to reduce PMN recruitment to and microvascular injury within the rodent lung following LPS exposure (80–82), likely due to dual effects upon PMN chemotaxis and endothelium. Statins also enhance clearance of apoptotic PMNs by alveolar macrophages (83). While statins do exert some effects on leukocyte function by depleting cellular cholesterol (84), other effects stem from depletion of cellular isoprenoids and consequent inhibition of cellular protein prenylation (80). Systemic treatment of rodents with synthetic agonists of Liver X Receptor (LXR), a nuclear receptor that promotes cellular cholesterol efflux and reverse cholesterol transport, has also been shown to reduce PMN recruitment into the airspace induced by LPS and Gram-negative bacteria (85, 86). A potential untoward consequence of reduced PMN recruitment to the lung through cholesterol targeting is impaired antibacterial host defense, as both statins and LXR agonists also reduce clearance of bacteria deposited in the rodent lung (80, 86). In the case of statins, impaired bacterial clearance may also reflect drug-induced defects in PMN bactericidal function (80). This said, clinical expression of pneumonia and its complications reflect not only pathogen proliferation, but also bystander tissue injury from responding host cells.
In recent years, the relationship between statin use and risk of community-acquired pneumonia has been examined in observational studies. Several retrospective studies have reported that statins are independently associated with either reduced risk of pneumonia (87, 88) or reduced pneumonia-associated mortality (89–93). Prospective, randomized, controlled trials will be necessary to resolve a potential role for statins in pneumonia therapy, however, as a prospective cohort study of patients hospitalized for community-acquired pneumonia reported no significant relationship between statins and a composite outcome of in-hospital mortality or admission to an intensive care unit (94). Given these findings, it may be difficult to conclusively rule out persistent confounding or a “healthy user” effect (95). Moreover, publication bias cannot be excluded.
The effect of cholesterol upon PMN recruitment to the lung likely depends upon the cell types having dysregulated cholesterol, the specific molecular features of the dyslipidemic state, and perhaps the nature of the alveolar or hematogenous insult. Contrary to the effects of localized pulmonary cholesterol overload observed in ABCG1 deficiency, systemic dyslipidemia induced by either diet or genetic manipulation is associated with reduced recruitment of PMNs to the LPS-exposed airspace, reflecting both reduced airspace chemokine expression and impaired PMN chemotaxis; dyslipidemic rodents nevertheless have increased airspace fluid protein, suggesting aggravated injury to the alveolocapillary barrier (M. B. Fessler, unpublished observations). Collectively, these data indicate that cholesterol has the potential to either promote or inhibit PMN recruitment to the airspaces, and may also impact lung injury through independent, direct effects on the integrity of the air–blood barrier. Given the high prevalence of dyslipidemia and its pharmacologic treatment in modern-day society, further investigation of the complex effects of cholesterol upon PMN recruitment to the lung is clearly needed.
Alcohol abuse has been associated with increased risk for bacterial pneumonia. Observational data show that alcoholic men and women suffer increased mortality from bacterial pneumonia compared with control subjects (96). A key element to early host defenses against invading microbes is the robust recruitment of neutrophils into the infected lung. Extensive work has shown conclusively shown that alcohol intoxication interferes with both the recruitment and functional capacity of neutrophils during bacterial infection of the lower respiratory tract (97–100).
The early expression of “alarm” cytokines, such as TNF-α, by alveolar macrophages is central to the development of the histologic hallmark of bacterial pneumonia: neutrophil infiltration (101, 102). Acute alcohol intoxication impairs lung TNF-α production in response to LPS at the post-transcriptional level (103, 104). By impairing the co-localization of TNF-α and the cell surface–bound TNF-α converting enzyme (TACE), acute ethanol exposure prevents cleavage of TNF-α from the producing cell's surface (105). The effect of chronic ethanol exposure on TNF-α release is controversial. While some work suggests chronic intoxication increases TNF-α release via augmented TACE activity (106) and stabilization of TNF-α mRNA (107), other studies show suppression of TNF-α production resulting from chronic alcohol (108, 109).
Although TNF-α is not itself a chemoattractant for neutrophils, it stimulates lung expression of CXC chemokines, which in turn promote the recruitment of neutrophils from the vasculature. Animal models of infection have shown that acute alcohol intoxication suppresses the lung's expression of the neutrophil chemokines MIP-2 and CINC, rodent orthologs of the human neutrophil chemokines IL-8 and Gro-α, which bind the CXCR2 receptor found on neutrophils (100, 110). This suppression is accompanied by decreased neutrophil recruitment, and exogenous chemokine administration has shown promise in restoring intrapulmonary neutrophil influx (111). The expression of neutrophil chemokines comes from many cell types in the lung, including the alveolar epithelium (112). Together, this suggests that alveolar parenchymal cells may be susceptible to alcohol. We recently investigated this hypothesis by examining the effect of acute intoxication on pulmonary expression of LIX, a neutrophil chemoattractant whose expression is limited to alveolar type II epithelial cells (113). Ethanol exposure decreased LIX expression in response to airway endotoxin challenge in vivo, and ethanol exposure down-regulated LIX in a dose-dependent manner in primary cultures of type II alveolar epithelium (114). These cells showed inhibition of NF-κB and p38 MAP kinase pathways in response to relatively low (25 mM) ethanol concentrations, suggesting that they are exquisitely sensitive targets of ethanol. In addition to the effects of ethanol on the lung epithelium, Zhang and coworkers have shown that acute ethanol intoxication profoundly suppresses the expression of the neutrophil chemokines S100A8 and S100A9 by leukocytes (115).
In addition to decreased chemokine expression, alcohol exposure results in intrinsic defects in the neutrophil that impede migration. During pneumonia, circulating neutrophils up-regulate the β2-integrin adhesion molecule, CD11b/CD18. CD11b/CD18 mediates neutrophil firm attachment to the pulmonary capillary endothelium and their subsequent transendothelial migration. Alcohol inhibits the up-regulation of CD18 expression on neutrophils in response to inflammatory stimuli (116) and suppresses neutrophil adhesion to the endothelium during appropriate stimulation (117). Furthermore, studies of neutrophils taken from chronic alcohol abusers show that these cells are hyporesponsive to chemotactic stimuli (118, 119). In alcoholics with liver disease, it has been proposed that systemic LPS exposure (through increased intestinal permeability and portal blood LPS content) induces a chronic inflammatory state. Neutrophil chemokines (IL-8) and chemotactic complement fragments (such as C5a) are elevated in the peripheral circulation of patients with alcoholic liver diseases (120, 121). The chronic in vivo activation of neutrophils has been postulated to account for the blunted response of neutrophils to chemoattractants in these hosts. Compounding this recruitment defect, alcohol-exposed neutrophils are less capable of killing bacteria owing to abnormalities in phagocytosis, degranulation, and superoxide generation (122, 123).
Alcohol-abusing patients are frequently leukopenic, and they often do not mount an appropriate leukocytosis during pneumonia (124). Given the importance of neutrophils in pathogen clearance, it is not surprising that neutropenia in alcoholics increases mortality from pulmonary infection, particularly bacteremic pneumococcal pneumonia (125). During bacterial pneumonia, the lung expresses G-CSF (126, 127). In contrast to most cytokines, which are “compartmentalized” within the lung during infection, G-CSF readily exits the pulmonary tissue and enters the circulation during infection (126, 128). G-CSF thus serves as a means of communication between the infected lung and bone marrow (129). Studies of systemic and pulmonary infection have shown that acute alcohol inhibits the expression of G-CSF (130), and animal studies have shown that recombinant G-CSF treatment can partially restore lung neutrophil recruitment in response to a bacterial stimulus (131). Examination of bone marrow from chronic alcoholics often shows hypocellularity, maturation arrest, and vacuolization of myeloid progenitor cells (132, 133). Bone marrow cells treated with clinically relevant ethanol concentrations show impaired granulocyte colony formation, also suggesting a direct toxic effect on these tissues (134, 135). In the context of bacterial infection, we have recently shown that alcohol inhibits the increase in granulopoietic progenitor cell proliferation in mice challenged with E. coli (136). This effect is at least partially due to alcohol preventing the phenotypic reversion of hematopoetic precursors to a less differentiated state, a phenomenon described in hematopoetic precursors (129) which appears requisite for optimal granulocyte production.
The discovery of IL-17 has broadened our understanding of how the lung solicits neutrophil influx during infection. A product of T lymphocytes and related cell types, IL-17 induces CXC chemokine production, which promotes neutrophilic inflammation (137). In animal studies, inoculation with K. pneumoniae induces a TLR4-dependent pulmonary IL-17 expression within 12 hours, and animals deficient in the receptor for IL-17 show enhanced mortality from infection (138). Chronic alcohol intoxication inhibits IL-17 secretion in the lungs in response to K. pneumoniae, and in vitro stimulation of T cells confirms a dose-dependent inhibition of IL-17 by ethanol (139). IL-23 is induced in alveolar macrophages and myeloid dendritic cells during challenge and is the dominant trigger for IL-17 (140). The expression of IL-23 in the lung during bacterial challenge is strongly suppressed by acute ethanol intoxication (55). By pretreating animals with an adenoviral vector encoding IL-17 prior to K. pneumoniae inoculation, neutrophil recruitment is restored and survival improved in alcohol-treated animals, suggesting that transient pharmacologic gene expression therapies may offer promise in the treatment of immunosuppressed patients with bacterial pneumonia (55). A product of NK T cells, invariant NK T (iNKT) cells, γ delta T cells, and CD4+ T cells, IL-17 induces CXC chemokine production, which promotes neutrophilic inflammation (58, 137, 141, 142). While the precise source(s) of lung IL-17 expression during acute bacterial infection are not known, work has shown that depletion of CD4+ or CD8+ cells results in a decrease in lung IL-17 expression as expressed during Klebsiella infection. It is likely that several of these cell populations each contribute to the IL-17 response to infection.
Bacterial pneumonia is an important disease. Neutrophils are one of the first cells to reach the site of bacterial infection. An ideal therapeutic strategy would be to attenuate the tissue-destructive potential of neutrophils without reducing their efficacy in antibacterial defense. Theoretically, one way to do this may be to identify therapeutic targets to attenuate excessive neutrophil accumulation during bacterial pneumonia. Our understanding of the molecular mechanisms that regulate neutrophil recruitment during infection/inflammation has improved substantially over recent years. Emerging studies indicate complex roles for cholesterol in neutrophil recruitment, and it has also been shown that alcohol affects neutrophil recruitment to the lungs during pneumonia. Although antibiotics are the rational treatment for pneumonias, antibiotic-resistant S. pneumoniae, H. influenzae, and S. aureus have been isolated from patients suffering from lower respiratory tract infections. The emergence of antibiotic-resistant pulmonary bacteria and the growing number of immunocompromised individuals have made the treatment of these infections increasingly difficult. The future challenge will be to apply our current understanding of neutrophil function to design therapeutic methods to maintain the host defense potential of neutrophils while modulating their destructive potential. In this context, therapeutic potential of TLRs and NLRs against bacterial infection in the lungs remains to be explored.
The authors are indebted to Scott Worthen for his support, guidance, and contribution to the work presented in this article. The authors thank Rachel Zemans, Ken Malcolm, Theivanthiran Balamayooran, and Kohila Mahadevan for critical reading of the manuscript.
This work was supported by a research grant from the American Lung Association (RG-22442-N to S.J.), a scientist award from the Flight Attendant Medical Research Institute (YCSA-062466 to S.J.). grants from the National Institutes of Health (R01 HL-091958 to S.J., K08 AA15163 to K.I.H.); and the Intramural Research Program of the NIH/NIEHS (to M.B.F.).
Originally Published in Press as DOI: 10.1165/rcmb.2009-0047TR on September 8, 2009
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.