SUMMARY Sepsis and the acute respiratory distress syndrome (ARDS) are major causes of mortality without targeted therapies. Although many experimental and clinical observations have implicated gut microbiota in the pathogenesis of these diseases, culture-based studies have failed to demonstrate translocation of bacteria to the lungs in critically ill patients. Here we report culture-independent evidence that the lung microbiome is enriched with gut bacteria both in a murine model of sepsis and in humans with established ARDS. Following experimental sepsis, lung communities were dominated by viable gut-associated bacteria. Ecologic analysis identified the lower gastrointestinal tract, rather than the upper respiratory tract, as the likely source community of post-sepsis lung bacteria. In bronchoalveolar lavage fluid from humans with ARDS, gut-specific bacteria (Bacteroides spp.) were common and abundant, undetected by culture, and correlated with the intensity of systemic inflammation. Alveolar TNF-α, a key mediator of alveolar inflammation in ARDS, was significantly correlated with altered lung microbiota. Our results demonstrate that the lung microbiome is enriched with gut-associated bacteria in sepsis and ARDS, potentially representing a shared mechanism of pathogenesis in these common and lethal diseases.

Sepsis results in a state of relative immunosuppression, rendering critically ill patients susceptible to secondary infections and increased mortality. Monocytes isolated from septic patients and experimental animals display a "deactivated" phenotype, characterized by impaired inflammatory and antimicrobial responses, including hyporesponsiveness to LPS. We investigated the role of the LPS/TLR4 axis and its inhibitor, IL-1 receptor-associated kinase-M (IRAK-M), in modulating the immunosuppression of sepsis using a murine model of peritonitis-induced sepsis followed by secondary challenge by intratracheal Pseudomonas aeruginosa. Septic mice demonstrated impaired alveolar macrophage function and increased mortality when challenged with intratracheal Pseudomonas as compared with nonseptic controls. TLR2 and TLR4 expression was unchanged in the lung following sepsis, whereas levels of IRAK-M were upregulated. Macrophages from IRAK-M-deficient septic mice produced higher levels of proinflammatory cytokines ex vivo and greater costimulatory molecule expression in vivo as compared with those of their WT counterparts. Following sepsis and secondary intrapulmonary bacterial challenge, IRAK-M -/-animals had higher survival rates and improved bacterial clearance from lung and blood compared with WT mice. In addition, increased pulmonary chemokine and inflammatory cytokine production was observed in IRAK-M -/-animals, leading to enhanced neutrophil recruitment to airspaces. Collectively, these findings indicate that IRAK-M mediates critical aspects of innate immunity that result in an immunocompromised state during sepsis.

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The contribution of neutrophils to lethal sensitivity and cytokine balance governing T1 and T2 host responses was assessed in a murine model of Legionella pneumophila pneumonia. Neutrophil depletion by administration of granulocyte-specific mAb RB6-8C5 at 1 day before infection rendered mice ∼100-fold more susceptible to lethal pneumonia induced by L. pneumophila. However, this treatment did not alter early bacterial clearance, despite a substantial decrease in neutrophil influx at this time point. Cytokine profiles in the lungs of control mice demonstrated strong T1 responses, characterized by an increase of IFN-γ and IL-12. In contrast, neutrophil-depleted mice exhibited significantly lower levels of IFN-γ and IL-12, and elevation of T2 cytokines, IL-4 and IL-10. Immunohistochemistry of bronchoalveolar lavage cells demonstrated the presence of IL-12 in neutrophils, but not alveolar macrophages. Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was paralleled to neutrophil number. However, intratracheal administration of recombinant murine IL-12 did not restore resistance, whereas reconstitution of IFN-γ drastically improved bacterial clearance and survival in neutrophil-depleted mice. Taken together, these data demonstrated that neutrophils play crucial roles in primary L. pneumophila infection, not via direct killing but more immunomodulatory effects. Our results suggest that the early recruitment of neutrophils may contribute to T1 polarization in a murine model of L. pneumophila pneumonia.

Leukocytes express both urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR, CD87). Evidence in vitro has implicated uPAR as a modulator of β2 integrin function, particularly CR3 (CD11b/CD18, Mac-1). Pseudomonas aeruginosa infection has been demonstrated to recruit neutrophils to the pulmonary parenchyma by a β2 integrin-dependent mechanism. We demonstrate that mice deficient in uPAR (uPAR−/−) have profoundly diminished neutrophil recruitment in response to P. aeruginosa pneumonia compared with wild-type (WT) mice. The requirement for uPAR in neutrophil recruitment is independent of the serine protease uPA, as neutrophil recruitment in uPA−/− mice is indistinguishable from recruitment in WT mice. uPAR−/− mice have impaired clearance of P. aeruginosa compared with WT mice, as demonstrated by CFU and comparative histology. WT mice have diminished neutrophil recruitment to the lung when an anti-CD11b mAb is given before inoculation with the pathogen, while recruitment of uPAR−/− neutrophils is unaffected. We conclude that uPAR is required for the recruitment of neutrophils to the lung in response to P. aeruginosa pneumonia and that this requirement is independent of uPA. Further, we show that uPAR and CR3 act by a common mechanism during neutrophil recruitment to the lung in response to P. aeruginosa. This is the first report of a requirement for uPAR during cellular recruitment in vivo against a clinically relevant pathogen.

We addressed the role of O⨪2 generated by the NADPH oxidase complex in the mechanism of polymorphonuclear leukocyte (PMN) accumulation and transalveolar migration and lung microvascular injury. Studies were made in mice lacking the p47phox and gp91phox subunits of NADPH oxidase (p47phox−/− and gp91phox−/−) in which PMN are incapable of the respiratory burst. The mice were challenged i.p. with live Escherichia coli to induce sepsis. We observed time-dependent increases in PMN sequestration and migration from 1 to 6 h after challenge with 2 × 108 E. coli. The responses in knockout mice were greater post-E. coli challenge compared with control mice; i.e., transalveolar PMN migration post-E. coli challenge increased by ∼50% in the null mice above values in wild type. The increased PMN infiltration was associated with decreased lung bacterial clearance. The generation of the chemoattractant macrophage-inflammatory protein-2 in lung tissue was greater in NADPH oxidase-defective mice after E. coli challenge than control mice; moreover, macrophage-inflammatory protein-2 Ab pretreatment prevented the PMN infiltration. We also observed that E. coli failed to increase lung microvascular permeability in p47phox−/− and gp91phox−/− mice despite the greater lung PMN sequestration. Thus, O⨪2 production is required for the induction of sepsis-induced lung microvascular injury. We conclude that NADPH oxidase-derived O⨪2 generation has an important bactericidal role, such that an impairment in bacterial clearance in NADPH oxidase-defective mice results in increased chemokine generation and lung tissue PMN infiltration.

Although cells of the innate inflammatory response, such as macrophages and neutrophils, have been extensively studied in the arena of Gram-negative bacterial pneumonia, a role for T cells remains unknown. To study the role of specific T cell populations in bacterial pneumonia, mice deleted of their TCR β- and/or δ-chain were intratracheally inoculated with Klebsiella pneumoniae. γδ T cell knockout mice displayed increased mortality at both early and late time points. In contrast, mice specifically lacking only αβ-T cells were no more susceptible than wild-type mice. Pulmonary bacterial clearance in γδ-T cell knockout mice was unimpaired. Interestingly, these mice displayed increased peripheral blood dissemination. Rapid up-regulation of IFN-γ and TNF-α gene expression, critical during bacterial infections, was markedly impaired in lung and liver tissue from γδ-T cell-deficient mice 24 h postinfection. The increased peripheral blood bacterial dissemination correlated with impaired hepatic bacterial clearance following pulmonary infection and increased hepatic injury as measured by plasma aspartate aminotransferase activity. Combined, these data suggest that mice lacking γδ-T cells have an impaired ability to resolve disseminated bacterial infections subsequent to the initial pulmonary infection. These data indicate that γδ-T cells comprise a critical component of the acute inflammatory response toward extracellular Gram-negative bacterial infections and are vital for the early production of the proinflammatory cytokines IFN-γ and TNF-α.

The roles of CXC chemokine-mediated host responses were examined with an A/J mouse model of Legionella pneumophila pneumonia. After intratracheal inoculation of 10 6 CFU of L. pneumophila, the bacterial numbers in the lungs increased 10-fold by day 2; this increase was accompanied by the massive accumulation of neutrophils. Reverse transcription-PCR data demonstrated the up-regulation of CXC chemokines, such as keratinocyte-derived chemokine, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Consistent with these data, increased levels of KC, MIP-2, and LIX proteins were observed in the lungs and peaked at days 1, 2, and 2, respectively. Although the administration of anti-KC or anti-MIP-2 antibody resulted in an approximately 20% decrease in neutrophil recruitment on day 2, no increase in mortality was observed. In contrast, the blockade of CXC chemokine receptor 2 (CXCR2), a receptor for CXC chemokines, including KC and MIP-2, strikingly enhanced mortality; this effect coincided with a 67% decrease in neutrophil recruitment. Interestingly, anti-CXCR2 antibody did not affect bacterial burden by day 2, even in the presence of a lethal challenge of bacteria. Moreover, a significant decrease in interleukin-12 (IL-12) levels, in contrast to the increases in KC, MIP-2, and LIX levels, was demonstrated for CXCR2-blocked mice. These data indicated that CXCR2-mediated neutrophil accumulation may play a crucial role in host defense against L. pneumophila pneumonia in mice. The increase in lethality without a change in early bacterial clearance suggested that neutrophils may exert their protective effect not through direct killing but through more immunomodulatory actions in L. pneumophila pneumonia. We speculate that a decrease in the levels of the protective cytokine IL-12 may explain, at least in part, the high mortality in the setting of reduced neutrophil recruitment.