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.

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.

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.

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.

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).

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).

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 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.