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Macrophages undergo profound metabolic reprogramming to join key immunoregulatory functions, which can be initiated by pattern recognition receptors. TREM2 (triggering receptor expressed on myeloid cells 2), a macrophage phagocytic receptor, plays pivotal roles in sepsis by enhancing bacterial clearance, which is associated with regulation of reactive oxygen species (ROS) production. However, how intracellular ROS participate in TREM2-mediated bactericidal activity remains unclear. This study was designed to investigate the organelle source and biological activity of ROS in the context of TREM2-mediated immune defense during Escherichia coli infection. Bone marrow-derived macrophages (BMDMs) were transfected with TREM2-overexpressing adenoviruses or control viruses and challenged with E. coli . The BMDMs were administered to mouse models with local E. coli infection. In addition, monocytic TREM2 expression, NOX2 concentrations, and pyroptosis were detected in patients with bacterial sepsis. General ROS production was found to be comparable between TREM2-overexpressing and control BMDMs upon E. coli challenge. The deficiency of Nox2 led to impaired phagosome degradation and lack of bactericidal ability and abolished TREM2-mediated protective activity against pulmonary E. coli infection. Overexpression of TREM2 suppressed mitochondrial ROS generation, inhibited NLRP3/caspase-1 inflammasome activation, and finally protected BMDMs from gasdermin D-mediated pyroptosis during pulmonary E. coli infection. The protective role of TREM2 was further confirmed in mice with abdominal E. coli infection. Moreover, monocytic TREM2 expression was positively correlated with NOX2 concentrations and negatively correlated with pyroptosis and disease severity in patients with bacterial sepsis. Collectively, TREM2 controls macrophage immune functions by fine-tuning ROS generation and enhances the host defense against bacterial infection. Our data suggest that TREM2 is a promising candidate target for sepsis therapy.

Resident immune cells (e.g., macrophages [MΦs]) and airway mucus clearance both contribute to a healthy lung environment. To investigate interactions between pulmonary MΦ function and defective mucus clearance, a genetic model of lysozyme M (LysM) promoter-mediated MΦ depletion was generated, characterized, and crossed with the sodium channel β subunit transgenic (Scnn1b-Tg) mouse model of defective mucus clearance. Diphtheria toxin A-mediated depletion of LysM(+) pulmonary MΦs in wild-type mice with normal mucus clearance resulted in lethal pneumonia in 24% of neonates. The pneumonias were dominated by Pasteurella pneumotropica and accompanied by emaciation, neutrophilic inflammation, and elevated Th1 cytokines. The incidence of emaciation and pneumonia reached 51% when LysM(+) MΦ depletion was superimposed on the airway mucus clearance defect of Scnn1b-Tg mice. In LysM(+) MΦ-depleted Scnn1b-Tg mice, pneumonias were associated with a broader spectrum of bacterial species and a significant reduction in airway mucus plugging. Bacterial burden (CFUs) was comparable between Scnn1b-Tg and nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice. However, the nonpneumonic LysM(+) MΦ-depleted Scnn1b-Tg mice exhibited increased airway inflammation, the presence of neutrophilic infiltration, and increased levels of inflammatory cytokines in bronchoalveolar lavage fluid compared with Scnn1b-Tg mice. Collectively, these data identify key MΦ-mucus clearance interactions with respect to both infectious and inflammatory components of muco-obstructive lung disease.

Triggering receptor expressed on myeloid cells (TREM)-1 and -2 can affect Toll-like receptor-mediated activation of immune cells. Klebsiella pneumoniae is a common cause of pneumonia-derived sepsis. Here we studied the role of TREM-1/3 and TREM-2 in the host response during Klebsiella pneumonia. Macrophages lacking either TREM-1/3 or TREM-2 were tested for their responsiveness toward K. pneumoniae and for their capacity to internalize this pathogen in vitro. TREM-1/3- and TREM-2-deficient mice were infected with K. pneumoniae via the airways, and their responses were compared with those in wild-type mice. TREM-1/3-deficient macrophages produced lower cytokine levels upon exposure to K. pneumoniae, whereas TREM-2-deficient macrophages released higher cytokine concentrations. TREM-2-deficient, but not TREM-1/3-deficient, macrophages showed a reduced capacity to phagocytose K. pneumoniae. TREM-1/3-deficient mice showed an impaired host defense during Klebsiella pneumonia, as reflected by worsened survival and increased bacterial growth and dissemination. In contrast, TREM-2 deficiency did not affect disease outcome. Although TREM-1/3 and TREM-2 influence macrophage responsiveness to K. pneumoniae in vitro, only TREM-1/3 contribute to the host response during Klebsiella pneumonia in vivo, serving a protective role.

The alveolar epithelium is composed of two cell types: type I cells comprise 95% of the gas exchange surface area, whereas type II cells secrete surfactant, while retaining the ability to convert into type I cells to induce alveolar repair. Using lineage-tracing analyses in the mouse model of Pseudomonas aeruginosa-induced lung injury, we identified a population of stem cell antigen (Sca)-1-expressing type II cells with progenitor cell properties that mediate alveolar repair. These cells were shown to be distinct from previously reported Sca-1-expressing bronchioalveolar stem cells. Microarray and Wnt reporter studies showed that surfactant protein (Sp)-C(+)Sca-1(+) cells expressed Wnt signaling pathway genes, and inhibiting Wnt/β-catenin signaling prevented the regenerative function of Sp-C(+)Sca-1(+) cells in vitro. Thus, P. aeruginosa-mediated lung injury induces the generation of a Sca-1(+) subset of type II cells. The progenitor phenotype of the Sp-C(+)Sca-1(+) cells that mediates alveolar epithelial repair might involve Wnt signaling.

Earlier studies indicated a role for reactive oxygen species (ROS) in host defense against Pseudomonas aeruginosa infection. However, the role of nicotinamide adenine dinucleotide phosphate-reduced (NADPH) oxidase (NOX) proteins and the mechanism of activation for NADPH oxidase in P. aeruginosa infection are not well-defined. Here, we investigated the role of NOX2 and NOX4 proteins in P. aeruginosa infection, ROS generation, and endothelial barrier function in murine lungs and in human lung microvascular endothelial cells (HLMVECs). Airway instillation of P. aeruginosa strain 103 (PA103) significantly increased ROS concentrations in bronchial alveolar lavage (BAL) fluid, along with the expression of NOX2 and NOX4, but not NOX1 and NOX3, in lung tissue. In addition, PA103-infected HLMVECs revealed elevated concentrations of ROS, NOX2, and NOX4. In murine lungs and HLMVECs, PA103 induced the NF-κB pathway, and its inhibition blocked PA103-dependent NOX2 and NOX4 expression. Barrier function analysis showed that heat-killed PA103 induced endothelial permeability in a dose-dependent manner, which was attenuated by treatment with small interfering (si)RNA specific for NOX4, but not NOX2. Furthermore, the knockdown of NOX4, but not NOX2, with siRNA reduced PA103-mediated apoptosis in HLMVECs. In vivo, the down-regulation of NOX4 with NOX4 siRNA attenuated PA103-induced lung vascular permeability. The deletion of NOX2 in mice exerted no effect on permeability, but offered significant resistance to P. aeruginosa-induced lung inflammation. These data show that P. aeruginosa lung infection up-regulates NOX2 and NOX4 expression and ROS generation, which play distinct roles in regulating lung inflammation, apoptosis, and permeability.

Proteoglycans (PGs) and their associated glycosaminoglycan side chains are effectors of inflammation, but little is known about changes to the composition of PGs in response to lung infection or injury. The goals of this study were to identify changes to heparan sulfate PGs in a mouse model of gram-negative pneumonia, to identify the Toll-like receptor adaptor molecules responsible for these changes, and to determine the role of the heparan sulfate PG in the innate immune response in the lungs. We treated mice with intratracheal LPS, a component of the cell wall of gram-negative bacteria, to model gram-negative pneumonia. Mice treated with intratracheal LPS had a rapid and selective increase in syndecan-4 mRNA that was regulated through MyD88-dependent mechanisms, whereas expression of several other PGs was not affected. To determine the role of syndecan-4 in the inflammatory response, we exposed mice deficient in syndecan-4 to LPS and found a significant increase in neutrophil numbers and amounts of CXC-chemokines and total protein in bronchoalveolar lavage fluid. In studies performed in vitro, macrophages and epithelial cells treated with LPS had increased expression of syndecan-4. Studies performed using BEAS-2B cells showed that pretreatment with heparin and syndecan-4 decreased the expression of CXCL8 mRNA in response to LPS and TNF-α. These findings indicate that the early inflammatory response to LPS involves marked up-regulation of syndecan-4, which functions to limit the extent of pulmonary inflammation and lung injury.

Rationale: After surviving the initial hyperinflammatory phase, patients with sepsis display features consistent with immunosuppression, which renders the host susceptible to nosocomial infections, in particular bacterial pneumonia. Suppression of tumorigenicity 2 (ST2) is a negative regulator of Toll-like receptor signaling implicated in endotoxin tolerance., Objectives: The present study sought to determine the role of ST2 in modulating host defense in the lung during sepsis, using a murine model of cecal ligation and puncture (CLP)-induced sepsis followed by a secondary infection with Pseudomonas aeruginosa via the airways., Methods: CLP or sham surgery was performed on BALB/c wild-type (WT) and ST2 knockout (KO) mice, and 24 hours later animals were challenged with 10(8) live P. aeruginosa., Measurements and Main Results: CLP mice demonstrated impaired clearance of Pseudomonas from their lungs and reduced pulmonary levels of tumor necrosis factor-α and IL-6 compared with sham mice. After CLP, ST2KO mice with secondary pneumonia displayed a strongly improved survival and a better bacterial clearance compared with WT mice, which was accompanied by enhanced lung inflammation. CLP did not influence the responsiveness of alveolar macrophages toward P. aeruginosa ex vivo irrespective of the st2 genotype. In contrast, CLP resulted in a reduced capacity of WT CD4(+) and CD8(+) T cells to produce IFN-γ and tumor necrosis factor-α, an immune suppressive effect that was not seen in ST2KO mice., Conclusions: These findings indicate that gene products of ST2 contribute to the immune-compromised state during sepsis and the ensuing disturbed homeostasis of lung host defense.

Elafin and secretory leukocyte protease inhibitor (SLPI) are pleiotropic molecules chiefly synthesized at the mucosal surface that have a fundamental role in the surveillance against microbial infections. Their initial discovery as anti-proteases present in the inflammatory milieu in chronic pathologies such as those of the lung suggested that they may play a role in keeping in check extracellular proteases released during the excessive activation of innate immune cells such as neutrophils. This soon proved to be a simplistic explanation, as other functions were also soon ascribed to these molecules (antimicrobial, modulation of innate and adaptive immunity, regulation of tissue repair). Data emanating from patients with chronic pathologies (in the lung and elsewhere) have shown that SLPI and elafin are often inactivated in inflammatory secretions, either through the action of host or microbial products, justifying attempts at antiprotease supplementation in clinical protocols. Although these have been sparse, proof of principle has been demonstrated, and future challenges will undoubtedly rest with improvements in methods of delivery in the context of tissue inflammation and in careful selection of patients more likely to benefit from SLPI/elafin augmentation.

MyD88 is an adapter protein required for the induction of proinflammatory cytokines by most Toll-like receptors (TLR), and Pseudomonas aeruginosa expresses ligands for multiple TLRs. MyD88(-/-) (KO) mice are highly susceptible to aerosolized P. aeruginosa, failing to elicit an early inflammatory response and permitting a 3-log increase in bacterial CFU in the lungs by 24 h after infection. We hypothesized that alveolar macrophages are the first cells to recognize and kill aerosolized P. aeruginosa in an MyD88-dependent fashion due to their location within the airways. To determine which cells in the lungs mediate MyD88-dependent defenses against P. aeruginosa, we generated radiation bone marrow (BM) chimeras between MyD88KO and wild-type (WT) mice. MyD88KO mice transplanted with MyD88KO BM (MyD88KO-->MyD88KO mice) displayed uncontrolled bacterial replication, whereas all other chimeras controlled the infection by 24 h. However, at 4 h, both MyD88KO-->MyD88KO and WT-->MyD88KO mice permitted intrapulmonary bacterial replication, whereas MyD88KO-->WT and WT-->WT mice did not, indicating that the source of BM had little impact on the early control of infection. Similarly, the genotype of the recipient rather than that of the BM donor determined early neutrophil recruitment to the lungs. Whereas intrapulmonary TNF-alpha and IL-1beta production were associated with WT BM, levels of the CXC chemokines MIP-2 and KC as well as GM-CSF were associated with recipient genotype. We conclude that lung parenchymal and BM-derived cells collaborate in the MyD88-dependent response to P. aeruginosa infection in the lungs in mice.

The virulence of Pseudomonas aeruginosa is partly controlled by the las quorum-sensing system. A rat model of acute pneumonia was used to investigate the pathophysiological impact of this system by comparing the virulence of the wild-type virulent laboratory strain PAO1 with that of its lasR-deleted mutant PAOR. In comparison with PAO1, PAOR was avirulent after an instillation of 106 cfu (mortality rates, 72 versus 0%, respectively; p < 0.0001). A ten-fold higher inoculum slightly increased the mortality rate induced by PAOR (25%), which remained lower than that induced by PAO1 (75%, p = 0.0001). In addition, with both inocula lung and bronchoalveolar lavage bacterial counts were significantly lower in rats infected with PAOR than with PAO1 (p

Pseudomonas aeruginosa is an opportunistic human pathogen that causes both an acute lung disease in patients with hospital-acquired pneumonia and a chronic lung disease in individuals with cystic fibrosis. Many of the pathophysiologic effects of P. aeruginosa infection are due to factors secreted by the bacterium. Conditioned media from cultures of P. aeruginosa increased interleukin-8 expression and decreased regulated on activation, normal T cells expressed and secreted (RANTES) expression by human airway epithelial cells. Both of these activities were present in heat-treated, protease-treated, small molecular weight fractions. The activities were not inhibited by polymyxin B and were not extracted into ethyl acetate, suggesting that they were not due to endotoxin or autoinducer. Conversely, results from chloroform extractions and studies with a phenazine-minus mutant suggested that the blue pigment pyocyanin contributes to these activities when present. In addition to the effects of small molecular weight factors on cytokine expression, proteases in bacterial-conditioned media further decreased levels of RANTES. By altering expression, release, and/or activity of inflammatory cytokines, secretory factors from P. aeruginosa could disrupt the delicate balance that constitutes the immune response to bacterial infection and thus could contribute to the lung damage that occurs in P. aeruginosa-infected airways.

Genetic factors are likely to contribute to the variable presentation of community-acquired pneumonia (CAP). The purpose of this prospective cohort study was to determine whether the LTalpha+250 (TNFbeta+250) and TNFalpha-308 gene polymorphisms are associated with different presentations of CAP. Septic shock (SS) was defined using American College of Chest Physicians/Society of Critical Care Medicine (ACCP-SCCM) criteria. Type I respiratory failure (T1RF) was defined as an O(2) saturation on room air of < 90% with a normal PCO(2). A total of 280 patients were genotyped; 31 had SS, 80 had T1RF. Genotype proportions are given in the order of AA/GA/ GG. The proportion of patients in each genotype developing SS was as follows: LTalpha+250 0.19/0.07/0.09 (p = 0.01 AA versus non-AA); TNFalpha-308 0.16/0.06/0.12 (p = NS). Carrying at least one AA (tumor necrosis factor [TNF] high secretor) genotype had an 18.0% risk of SS versus 6.8% (p = 0.006). GG homozygotes (TNF low secretors) at both loci had only a 2.9% risk of SS. Septic shock was associated with the LTalpha+250:TNFalpha-308 A:G haplotype but not the A:A haplotype, suggesting that LTalpha+250 is a marker, rather than a causative polymorphism. Carriage of the G:G haplotype had a significant protective effect against the development of septic shock (p = 0.011). T1RF was not associated with LTalpha+250 AA genotype. In the absence of septic shock, there was a significant trend to greater T1RF in patients with LTalpha+250 GG (TNFalpha hyposecretor) genotype (p = 0.03). Our finding of different genotype associations for SS and T1RF has important implications for immunotherapy in both CAP and sepsis, as well as for the definition of the systemic inflammatory response syndrome (SIRS).