Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

Staphylococcus aureus stimulates neutrophil itaconate production that suppresses the oxidative burst.

Neutrophils are critical in the host defense against Staphylococcus aureus, a major human pathogen. However, even in the setting of a robust neutrophil response, S. aureus can evade immune clearance. Here, we demonstrate that S. aureus impairs neutrophil function by triggering the production of the anti-inflammatory metabolite itaconate. The enzyme that synthesizes itaconate, Irg1, is selectively expressed in neutrophils during S. aureus pneumonia. Itaconate inhibits neutrophil glycolysis and oxidative burst, which impairs survival and bacterial killing. In a murine pneumonia model, neutrophil Irg1 expression protects the lung from excessive inflammation but compromises bacterial clearance. S. aureus is thus able to evade the innate immune response by targeting neutrophil metabolism and inducing the production of the anti-inflammatory metabolite itaconate.

Immunometabolites Drive Bacterial Adaptation to the Airway.

Pseudomonas aeruginosa and Staphylococcus aureus are both opportunistic pathogens that are frequently associated with chronic lung infections. While bacterial virulence determinants are critical in initiating infection, the metabolic flexibility of these bacteria promotes their persistence in the airway. Upon infection, these pathogens induce host immunometabolic reprogramming, resulting in an airway milieu replete with immune-signaling metabolites. These metabolites are often toxic to the bacteria and create a steep selection pressure for the emergence of bacterial isolates adapted for long-term survival in the inflamed lung. In this review, we discuss the main differences in the host immunometabolic response to P. aeruginosa and S. aureus, as well as how these pathogens alter their own metabolism to adapt to airway metabolites and cause persistent lung infections.

Staphylococcus aureus induces an itaconate-dominated immunometabolic response that drives biofilm formation.

Staphylococcus aureus is a prominent human pathogen that readily adapts to host immune defenses. Here, we show that, in contrast to Gram-negative pathogens, S. aureus induces a distinct airway immunometabolic response dominated by the release of the electrophilic metabolite, itaconate. The itaconate synthetic enzyme, IRG1, is activated by host mitochondrial stress, which is induced by staphylococcal glycolysis. Itaconate inhibits S. aureus glycolysis and selects for strains that re-direct carbon flux to fuel extracellular polysaccharide (EPS) synthesis and biofilm formation. Itaconate-adapted strains, as illustrated by S. aureus isolates from chronic airway infection, exhibit decreased glycolytic activity, high EPS production, and proficient biofilm formation even before itaconate stimulation. S. aureus thus adapts to the itaconate-dominated immunometabolic response by producing biofilms, which are associated with chronic infection of the human airway.

Hemozoin Promotes Lung Inflammation via Host Epithelial Activation.

Respiratory distress in severe malaria is associated with high mortality, but its pathogenesis remains unclear. The malaria pigment hemozoin (HZ) is abundant in target organs of severe malaria, including the lungs, and is known to be a potent innate immune activator of phagocytes. We hypothesized that HZ might also stimulate lung epithelial activation and thereby potentiate lung inflammation. We show here that airway epithelium stimulated with HZ undergoes global transcriptional reprogramming and changes in cell surface protein expression that comprise an epithelial activation phenotype. Proinflammatory signaling is induced, and key cytoadherence molecules are upregulated, including several associated with severe malaria, such as CD36 and ICAM1. Epithelial and extracellular matrix remodeling pathways are transformed, including induction of key metalloproteases and modulation of epithelial junctions. The overall program induced by HZ serves to promote inflammation and neutrophil transmigration, and is recapitulated in a murine model of HZ-induced acute pneumonitis. Together, our data demonstrate a direct role for hemozoin in stimulating epithelial activation that could potentiate lung inflammation in malaria.IMPORTANCE Respiratory distress (RD) is a complication of severe malaria associated with a particularly high risk for death in African children infected with the parasite Plasmodium falciparum The pathophysiology underlying RD remains poorly understood, and the condition is managed supportively. The parasite-derived factor HZ accumulates in target organs of severe malaria, including the lungs, and is a potent stimulator of immune cells. Our findings demonstrate that HZ causes global activation of lung epithelial cells, a response that directly promotes lung inflammation. HZ stimulates expression of key proinflammatory and cell surface molecules, alters signaling pathways involved in epithelial-matrix remodeling, and promotes neutrophil transmigration and airway inflammation. The lung epithelial activation induced by HZ mimics patterns seen in malarial lung injury and provides new insights into the molecular pathogenesis of RD.

Future Research Directions in Pneumonia. NHLBI Working Group Report.

Pneumonia is a complex pulmonary disease in need of new clinical approaches. Although triggered by a pathogen, pneumonia often results from dysregulations of host defense that likely precede infection. The coordinated activities of immune resistance and tissue resilience then dictate whether and how pneumonia progresses or resolves. Inadequate or inappropriate host responses lead to more severe outcomes such as acute respiratory distress syndrome and to organ dysfunction beyond the lungs and over extended time frames after pathogen clearance, some of which increase the risk for subsequent pneumonia. Improved understanding of such host responses will guide the development of novel approaches for preventing and curing pneumonia and for mitigating the subsequent pulmonary and extrapulmonary complications of pneumonia. The NHLBI assembled a working group of extramural investigators to prioritize avenues of host-directed pneumonia research that should yield novel approaches for interrupting the cycle of unhealthy decline caused by pneumonia. This report summarizes the working group's specific recommendations in the areas of pneumonia susceptibility, host response, and consequences. Overarching goals include the development of more host-focused clinical approaches for preventing and treating pneumonia, the generation of predictive tools (for pneumonia occurrence, severity, and outcome), and the elucidation of mechanisms mediating immune resistance and tissue resilience in the lung. Specific areas of research are highlighted as especially promising for making advances against pneumonia.

Disruption of staphylococcal aggregation protects against lethal lung injury.

Infection by Staphylococcus aureus strain USA300 causes tissue injury, multiorgan failure, and high mortality. However, the mechanisms by which the bacteria adhere to, then stabilize on, mucosal surfaces before causing injury remain unclear. We addressed these issues through the first real-time determinations of USA300-alveolar interactions in live lungs. We found that within minutes, inhaled USA300 established stable, self-associated microaggregates in niches at curved, but not at flat, regions of the alveolar wall. The microaggregates released α-hemolysin toxin, causing localized alveolar injury, as indicated by epithelial dye loss, mitochondrial depolarization, and cytosolic Ca2+ increase. Spread of cytosolic Ca2+ through intercellular gap junctions to adjoining, uninfected alveoli caused pulmonary edema. Systemic pretreatment with vancomycin, a USA300-cidal antibiotic, failed to protect mice infected with inhaled WT USA300. However, vancomycin pretreatment markedly abrogated mortality in mice infected with mutant USA300 that lacked the aggregation-promoting factor PhnD. We interpret USA300-induced mortality as having resulted from rapid bacterial aggregation in alveolar niches. These findings indicate, for the first time to our knowledge, that alveolar microanatomy is critical in promoting the aggregation and, hence, in causing USA300-induced alveolar injury. We propose that in addition to antibiotics, strategies for bacterial disaggregation may constitute novel therapy against USA300-induced lung injury.

Lambda Interferon Restructures the Nasal Microbiome and Increases Susceptibility to Staphylococcus aureus Superinfection.

UNLABELLED: Much of the morbidity and mortality associated with influenza virus respiratory infection is due to bacterial coinfection with pathogens that colonize the upper respiratory tract such as methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae. A major component of the immune response to influenza virus is the production of type I and III interferons. Here we show that the immune response to infection with influenza virus causes an increase and restructuring of the upper respiratory microbiota in wild-type (WT) mice but not in Il28r(-/-) mutant mice lacking the receptor for type III interferon. Mice lacking the IL-28 receptor fail to induce STAT1 phosphorylation and expression of its regulator, SOCS1. Il28r(-/-) mutant mice have increased expression of interleukin-22 (IL-22), as well as Ngal and RegIIIγ, in the nasal cavity, the source of organisms that would be aspirated to cause pneumonia. Proteomic analysis reveals changes in several cytoskeletal proteins that contribute to barrier function in the nasal epithelium that may contribute to the effects of IL-28 signaling on the microbiota. The importance of the effects of IL-28 signaling in the pathogenesis of MRSA pneumonia after influenza virus infection was confirmed by showing that WT mice nasally colonized before or after influenza virus infection had significantly higher levels of infection in the upper airways, as well as significantly greater susceptibility to MRSA pneumonia than Il28r(-/-) mutant mice did. Our results suggest that activation of the type III interferon in response to influenza virus infection has a major effect in expanding the upper airway microbiome and increasing susceptibility to lower respiratory tract infection. IMPORTANCE: S. aureus and influenza virus are important respiratory pathogens, and coinfection with these organisms is associated with significant morbidity and mortality. The ability of influenza virus to increase susceptibility to S. aureus infection is less well understood. We show here that influenza virus leads to a change in the upper airway microbiome in a type III interferon-dependent manner. Mice lacking the type III interferon receptor have altered STAT1 and IL-22 signaling. In coinfection studies, mice without the type III interferon receptor had significantly less nasal S. aureus colonization and subsequent pneumonia than infected WT mice did. This work demonstrates that type III interferons induced by influenza virus contribute to nasal colonization and pneumonia due to S. aureus superinfection.

Rational manipulation of mRNA folding free energy allows rheostat control of pneumolysin production by Streptococcus pneumoniae.

The contribution of specific factors to bacterial virulence is generally investigated through creation of genetic "knockouts" that are then compared to wild-type strains or complemented mutants. This paradigm is useful to understand the effect of presence vs. absence of a specific gene product but cannot account for concentration-dependent effects, such as may occur with some bacterial toxins. In order to assess threshold and dose-response effects of virulence factors, robust systems for tunable expression are required. Recent evidence suggests that the folding free energy (ΔG) of the 5' end of mRNA transcripts can have a significant effect on translation efficiency and overall protein abundance. Here we demonstrate that rational alteration of 5' mRNA folding free energy by introduction of synonymous mutations allows for predictable changes in pneumolysin (PLY) expression by Streptococcus pneumoniae without the need for chemical inducers or heterologous promoters. We created a panel of isogenic S. pneumoniae strains, differing only in synonymous (silent) mutations at the 5' end of the PLY mRNA that are predicted to alter ΔG. Such manipulation allows rheostat-like control of PLY production and alters the cytotoxicity of whole S. pneumoniae on primary and immortalized human cells. These studies provide proof-of-principle for further investigation of mRNA ΔG manipulation as a tool in studies of bacterial pathogenesis.

CD4+ T cells promote the pathogenesis of Staphylococcus aureus pneumonia.

We postulated that the activation of proinflammatory signaling by methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 is a major factor in the pathogenesis of severe pneumonia and a target for immunomodulation. Local activation of T cells in the lung was a conserved feature of multiple strains of S. aureus, in addition to USA300. The pattern of Vß chain activation was consistent with known superantigens, but deletion of SelX or SEK and SEQ was not sufficient to prevent T-cell activation, indicating the participation of multiple genes. Using Rag2(-/-), Cd4(-/-), and Cd28(-/-) mice, we observed significantly improved clearance of MRSA from the airways and decreased lung pathology, compared with findings for wild-type controls. The improved outcome correlated with decreased production of proinflammatory cytokines (tumor necrosis factor, KC, interleukin 6, and interleukin 1ß). Our data suggest that T-cell-mediated hypercytokinemia induced by infection with MRSA strain USA300 contributes to pathogenesis and may be a therapeutic target for improving outcomes of this common infection in a clinical setting.

Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity.

The tissue-resident macrophages of barrier organs constitute the first line of defence against pathogens at the systemic interface with the ambient environment. In the lung, resident alveolar macrophages (AMs) provide a sentinel function against inhaled pathogens. Bacterial constituents ligate Toll-like receptors (TLRs) on AMs, causing AMs to secrete proinflammatory cytokines that activate alveolar epithelial receptors, leading to recruitment of neutrophils that engulf pathogens. Because the AM-induced response could itself cause tissue injury, it is unclear how AMs modulate the response to prevent injury. Here, using real-time alveolar imaging in situ, we show that a subset of AMs attached to the alveolar wall form connexin 43 (Cx43)-containing gap junction channels with the epithelium. During lipopolysaccharide-induced inflammation, the AMs remained sessile and attached to the alveoli, and they established intercommunication through synchronized Ca(2+) waves, using the epithelium as the conducting pathway. The intercommunication was immunosuppressive, involving Ca(2+)-dependent activation of Akt, because AM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretion of proinflammatory cytokines in the bronchoalveolar lavage. A picture emerges of a novel immunomodulatory process in which a subset of alveolus-attached AMs intercommunicates immunosuppressive signals to reduce endotoxin-induced lung inflammation.

Bacterial pathogens activate a common inflammatory pathway through IFNλ regulation of PDCD4.

The type III interferon (IFNλ) receptor IL-28R is abundantly expressed in the respiratory tract and has been shown essential for host defense against some viral pathogens, however no data are available concerning its role in the innate immune response to bacterial pathogens. Staphylococcus aureus and Pseudomonas aeruginosa induced significant production of IFNλ in the lung, and clearance of these bacteria from the lung was significantly increased in IL-28R null mice compared to controls. Improved bacterial clearance correlated with reduced lung pathology and a reduced ratio of pro- vs anti-inflammatory cytokines in the airway. In human epithelial cells IFNλ inhibited miR-21 via STAT3 resulting in upregulation of PDCD4, a protein known to promote inflammatory signaling. In vivo 18 hours following infection with either pathogen, miR-21 was significantly reduced and PDCD4 increased in the lungs of wild type compared to IL-28R null mice. Infection of PDCD4 null mice with USA300 resulted in improved clearance, reduced pathology, and reduced inflammatory cytokine production. These data suggest that during bacterial pneumonia IFNλ promotes inflammation by inhibiting miR-21 regulation of PDCD4.

Activation of inflammasome signaling mediates pathology of acute P. aeruginosa pneumonia.

The respiratory tract is exceptionally well defended against infection from inhaled bacteria, with multiple proinflammatory signaling cascades recruiting phagocytes to clear airway pathogens. However, organisms that efficiently activate damaging innate immune responses, such as those mediated by the inflammasome and caspase-1, may cause pulmonary damage and interfere with bacterial clearance. The extracellular, opportunistic pathogen Pseudomonas aeruginosa expresses not only pathogen-associated molecular patterns that activate NF-κB signaling in epithelial and immune cells, but also flagella that activate the NLRC4 inflammasome. We demonstrate that induction of inflammasome signaling, ascribed primarily to the alveolar macrophage, impaired P. aeruginosa clearance and was associated with increased apoptosis/pyroptosis and mortality in a murine model of acute pneumonia. Strategies that limited inflammasome activation, including infection by fliC mutants, depletion of macrophages, deletion of NLRC4, reduction of IL-1ß and IL-18 production, inhibition of caspase-1, and inhibition of downstream signaling in IL-1R- or IL-18R-null mice, all resulted in enhanced bacterial clearance and diminished pathology. These results demonstrate that the inflammasome provides a potential target to limit the pathological consequences of acute P. aeruginosa pulmonary infection.

Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity.

Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments.

The length of the Staphylococcus aureus protein A polymorphic region regulates inflammation: impact on acute and chronic infection.

Staphylococcus aureus protein A (SpA) plays a critical role in the induction of inflammation. This study was aimed to determine whether the number of short sequence repeats (SSRs) present in the polymorphic region modulates the inflammatory response induced by SpA. We demonstrated that there is a dose-response effect in the activation of interferon (IFN)-ß signaling in airway epithelial and immune cells, depending on the number of SSRs, which leads to differences in neutrophil recruitment. We also determined that a significant proportion of isolates from patients with chronic infections such as osteomyelitis and cystic fibrosis carry fewer SSRs than do isolates from patients with acute infections or healthy carriers and that there was an inverse correlation between the number of SSRs and the length of disease course. Given the importance of IFN signaling in eradication of S. aureus, loss of SSRs may represent an advantageous mechanism to adapt to and persist in the host.

TLR2 regulates gap junction intercellular communication in airway cells.

The innate immune response to inhaled bacteria, such as the opportunist Pseudomonas aeruginosa, is initiated by TLR2 displayed on the apical surface of airway epithelial cells. Activation of TLR2 is accompanied by an immediate Ca(2+) flux that is both necessary and sufficient to stimulate NF-kappaB and MAPK proinflammatory signaling to recruit and activate polymorphonuclear leukocytes in the airway. In human airway cells, gap junction channels were found to provide a regulated conduit for the movement of Ca(2+) from cell to cell. In response to TLR2 stimulation, by either lipid agonists or P. aeruginosa, gap junctions functioned to transiently amplify proinflammatory signaling by communicating Ca(2+) fluxes from stimulated to adjacent, nonstimulated cells thus increasing epithelial CXCL8 production. P. aeruginosa stimulation also induced tyrosine phosphorylation of connexin 43 and association with c-Src, events linked to the closure of these channels. By 4 h postbacterial stimulation, gap junction communication was decreased indicating an autoregulatory control of the connexins. Thus, gap junction channels comprised of connexin 43 and other connexins in airway cells provide a mechanism to coordinate and regulate the epithelial immune response even in the absence of signals from the immune system.

The type III toxins of Pseudomonas aeruginosa disrupt epithelial barrier function.

The type III secreted toxins of Pseudomonas aeruginosa are important virulence factors associated with clinically important infection. However, their effects on bacterial invasion across mucosal surfaces have not been well characterized. One of the most commonly expressed toxins, ExoS, has two domains that are predicted to affect cytoskeletal integrity, including a GTPase-activating protein (GAP) domain, which targets Rho, a major regulator of actin polymerization; and an ADP-ribosylating domain that affects the ERM proteins, which link the plasma membrane to the actin cytoskeleton. The activities of these toxins, and ExoS specifically, on the permeability properties of polarized airway epithelial cells with intact tight junctions were examined. Strains expressing type III toxins altered the distribution of the tight junction proteins ZO-1 and occludin and were able to transmigrate across polarized airway epithelial monolayers, in contrast to DeltaSTY mutants. These effects on epithelial permeability were associated with the ADP-ribosylating domain of ExoS, as bacteria expressing plasmids lacking expression of the ExoS GAP activity nonetheless increased the permeation of fluorescent dextrans, as well as bacteria, across polarized airway epithelial cells. Treatment of epithelial cells with cytochalasin D depolymerized actin filaments and increased permeation across the monolayers but did not eliminate the differential effects of wild-type and toxin-negative mutants on the epithelial cells, suggesting that additional epithelial targets are involved. Confocal imaging studies demonstrated that ZO-1, occludin, and ezrin undergo substantial redistribution in human airway cells intoxicated by ExoS, -T, and -Y. These studies support the hypothesis that type III toxins enhance P. aeruginosa's invasive capabilities by interacting with multiple eukaryotic cytoskeletal components.

Staphylococcus aureus protein A activates TACE through EGFR-dependent signaling.

Among the many adhesins and toxins expressed by Staphylococcus aureus, protein A is an exceptionally complex virulence factor, known to interact with multiple eukaryotic targets, particularly those with immunological functions. Protein A acts as a ligand that can mimic TNF-alpha to activate TNFR1 and subsequent proinflammatory signaling. It also stimulates the cleavage of TNFR1 from the surface of epithelial cells and macrophages, which serves to limit TNF-alpha signaling. We characterized the signaling pathway responsible for TNFR1 shedding and identified protein A mutants which could activate TNFR1-dependent signaling, but were unable to activate TACE, the TNFR1 sheddase. Activation of TACE was dependent upon a discrete interaction between the previously defined IgG-binding domain of protein A and the epidermal growth factor receptor (EGFR), which in turn induced TACE phosphorylation through a c-Src-erk1/2-mediated cascade. This novel interaction was independent of the autocrine activation of EGFR and protein A-induced TGF-alpha was neither required nor sufficient to activate TNFR1 shedding. Thus, staphylococci exploit the ubiquitous and multifunctional EGFR to regulate the availability of TNFR1 on mucosal and immune cells.

Staphylococcus aureus protein A activates TNFR1 signaling through conserved IgG binding domains.

Staphylococcus aureus continues to be a major cause of infection in normal as well as immunocompromised hosts, and the increasing prevalence of highly virulent community-acquired methicillin-resistant strains is a public health concern. A highly expressed surface component of S. aureus, protein A (SpA), contributes to its success as a pathogen by both activating inflammation and by interfering with immune clearance. SpA is known to bind to IgG Fc, which impedes phagocytosis. SpA is also a potent activator of tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) signaling, inducing both chemokine expression and TNF-converting enzyme-dependent soluble TNFR1 (sTNFR1) shedding, which has anti-inflammatory consequences, particularly in the lung. Using a collection of glutathione S-transferase fusions to the intact IgG binding region of SpA and to each of the individual binding domains, we found that the SpA IgG binding domains also mediate binding to human airway cells. TNFR1-dependent CXCL8 production could be elicited by any one of the individual SpA IgG binding domains as efficiently as by either the entire SpA or the intact IgG binding region. SpA induction of sTNFR1 shedding required the entire IgG binding region and tolerated fewer substitutions in residues known to interact with IgG. Each of the repeated domains of the IgG binding domain can affect multiple immune responses independently, activating inflammation through TNFR1 and thwarting opsonization by trapping IgG Fc domains, while the intact IgG binding region can limit further signaling through sTNFR1 shedding.

Cell signaling underlying the pathophysiology of pneumonia.

The symposium addressed the burgeoning interest in fundamental mechanisms underlying the onset of pneumonia. Bacteria exploit the lung's innate immune mechanism, resulting in pathophysiological cell signaling. As a consequence inflammation develops, leading to pneumonia. New mechanisms have been identified by which bacteria or bacterial products in the airway induce cross-compartmental signaling that leads to inflammatory consequences. The speakers addressed activation of the transcription factor, NF-kappaB occurring as a consequence of bacterial interactions with specific receptors, such as the Toll-like receptors and the TNF receptor 1 (Prince), or as a consequence of cytokine induction (Mizgerd). Also considered were mechanisms of bacterial virulence in the clinical setting (Wiener-Kronish) and the role of alveolar-capillary signaling mechanisms in the initiation of lung inflammation.