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Background Hidradenitis suppurativa (HS) is a chronic inflammatory and debilitating disease of the skin. No biomarkers for this disease exist. Objectives We set out to test whether angiotensin-converting enzyme (ACE), lysozyme, soluble interleukin 2 receptor (sIL-2R) and S100A8 ⁄A9 (calprotectin) are elevated in patients with HS. Methods Serum was collected from 29 patients with HS at different stages of the disease, and from 51 controls. ACE, lysozyme, sIL-2R and S100A8 ⁄A9 levels were measured. Clinical observation of disease activity was scored according to the Hurley grading system and by a physician global score (PGS) of disease severity. Results Serum levels of lysozyme and ACE were not increased above the normal reference values in controls or patients with HS. Levels of sIL-2R and S100A8 ⁄A9 were significantly higher in patients with HS than in controls (P

Mechanical ventilation (MV) has the potential to induce lung damage in healthy lungs or aggravate existing lung injury. Polymorphonuclear neutrophil (PMN) recruitment plays an important role in driving the inflammatory response in ventilator-induced lung injury (VILI). The cyclin-dependent kinase inhibitor r-roscovitine has been shown to induce apoptosis in PMNs. In this study, we investigated the potential of r-roscovitine treatment in reducing lung damage in a mouse model of VILI. Mice were tracheotomized and subjected to lung-protective MV with lower (similar to 7.5 mL/kg) or lung-injurious MV with higher (similar to 15 mL/kg) tidal volume (V-T). R-roscovitine treatment enhanced apoptosis in PMNs in vitro. Ventilator-induced lung injury was associated with pulmonary PMN influx in low and high V-T MV. During lung-injurious MV, r-roscovitine treatment reduced the number of PMNs and lowered levels of the lung damage markers RAGE (receptor for advanced glycation end products) and total immunoglobulin M in bronchoalveolar lavage fluid. R-roscovitine did not affect cytokine or chemokine levels in the bronchoalveolar space, neither during lung-protective nor lung-injurious MV. Thus, r-roscovitine treatment reduces lung damage in VILI, possibly dependent on increased apoptosis of PMNs

Bacterial pneumonia remains associated with high morbidity and mortality. The gram-positive pathogen Streptococcus pneumoniae is the most common cause of community-acquired pneumonia. Lipoteichoic acid (LTA) is an important proinflammatory component of the gram-positive bacterial cell wall. R-roscovitine, a purine analog, is a potent cyclin-dependent kinase (CDK)-1, -2, -5 and -7 inhibitor that has the ability to inhibit the cell cycle and to induce polymorphonuclear cell (PMN) apoptosis. We sought to investigate the effect of R-roscovitine on LTA-induced activation of cell lines with relevance for lung inflammation in vitro and on lung inflammation elicited by either LTA or viable S. pneumoniae in vivo. In vitro R-roscovitine enhanced apoptosis in PMNs and reduced tumor necrosis factor (TNF)-alpha and keratinocyte chemoattractant (KC) production in MH-S (alveolar macrophage) and MLE-12/MLE-15 (respiratory epithelial) cell lines. In vivo R-roscovitine treatment reduced PMN numbers in bronchoalveolar lavage fluid during LTA-induced lung inflammation; this effect was reversed by inhibiting apoptosis. Postponed treatment with R-roscovitine (24 and 72 h) diminished PMN numbers in lung tissue during gram-positive pneumonia; this step was associated with a transient increase in pulmonary bacterial loads. R-roscovitine inhibits proinflammatory responses induced by the gram-positive stimuli LTA and S. pneumoniae. R-roscovitine reduces PMN numbers in lungs upon LTA administration by enhancing apoptosis. The reduction in PMN numbers caused by R-roscovitine during S. pneumoniae pneumonia may hamper antibacterial defense. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00033

Background The innate immune response is important in ventilator-induced lung injury (VILI) but the exact pathways involved are not elucidated. The authors studied the role of the intracellular danger sensor NLRP3 inflammasome. Methods NLRP3 inflammasome gene expression was analyzed in respiratory epithelial cells and alveolar macrophages obtained from ventilated patients (n = 40). In addition, wild-type and NLRP3 inflammasome deficient mice were randomized to low tidal volume (approximately 7.5 ml/kg) and high tidal volume (approximately 15 ml/kg) ventilation. The presence of uric acid in lung lavage, activation of caspase-1, and NLRP3 inflammasome gene expression in lung tissue were investigated. Moreover, mice were pretreated with interleukin-1 receptor antagonist, glibenclamide, or vehicle before start of mechanical ventilation. VILI endpoints were relative lung weights, total protein in lavage fluid, neutrophil influx, and pulmonary and systemic cytokine and chemokine concentrations. Data represent mean ± SD. Results Mechanical ventilation up-regulated messenger RNA expression levels of NLRP3 in alveolar macrophages (1.0 ± 0 vs. 1.70 ± 1.65, P less than 0.05). In mice, mechanical ventilation increased both NLRP3 and apoptosis-associated speck-like protein messenger RNA levels, respectively (1.08 ± 0.55 vs. 3.98 ± 2.89; P less than 0.001 and 0.95 ± 0.53 vs. 6.0 ± 3.55; P less than 0.001), activated caspase-1, and increased uric acid levels (6.36 ± 1.85 vs. 41.9 ± 32.0, P less than 0.001). NLRP3 inflammasome deficient mice displayed less VILI due to high tidal volume mechanical ventilation compared with wild-type mice. Furthermore, treatment with interleukin-1 receptor antagonist or glibenclamide reduced VILI. Conclusions Mechanical ventilation induced a NLRP3 inflammasome dependent pulmonary inflammatory response. NLRP3 inflammasome deficiency partially protected mice from VILI.

Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential to immune cells. The current study explored signal transduction events during murine Gram-negative pneumonia using a systems biology approach. Kinase activity arrays enable the analysis of 1,024 consensus sequences of protein kinase substrates. Using a kinase activity array on whole lung lysates, cellular kinase activities were determined in a mouse model of K. pneumoniae pneumonia. Notable kinase activities also were validated with phospho-specific Western blots. On the basis of the profiling data, mitogen-activated protein kinase (MAPK) signaling via p42 mitogen-activated protein kinase (p42) and p38 mitogen-activated protein kinase (p38) and transforming growth factor beta (TGF beta) activity were reduced during infection, whereas v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) activity generally was enhanced. AKT signaling was represented in both metabolic and inflammatory (mitogen-activated protein kinase kinase 2 (MKK), apoptosis signal-regulating kinase/mitogen-activated protein kinase kinase kinase 5 (ASK) and v-raf murine sarcoma viral oncogene homolog B1 (b-RAF)) context. This study reaffirms the importance of classic inflammation pathways, such as MAPK and TGF beta signaling and reveals less known involvement of glycogen synthase kinase 3 beta (GSK-3 beta), AKT and SRC signaling cassettes in pneumonia. (C) 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.org

The activation of type 3 deiodinase (D3) has been postulated to play a role in the reduction of thyroid hormone levels during illness. Using a mouse model of acute bacterial infection, we have recently demonstrated marked D3 immunostaining in neutrophils infiltrating infected organs. These observations suggest a possible additional role for this enzyme in the innate immune response. To further assess the role of D3 in the response to acute bacterial infection, we used null D3 [D3 knockout (D3KO)] and wild type (WT) mice and infected them with Streptococcus pneumoniae. Marked reductions in serum thyroid hormone levels were observed both in D3KO and WT mice. Infection resulted also in a decrease in liver D1 activity in WT, but not in infected D3KO mice. Upon infection, pulmonary neutrophilic influx (measured by myeloperoxidase levels) and IL-6 and TNF concentrations increased equally in D3KO and WT mice, and histological examination of infected mice showed similar pulmonary inflammation in both strains. However, D3KO animals demonstrated significantly higher bacterial load in blood, lung, and spleen compared with WT mice. We conclude that 1) D3 is not required to generate the systemic manifestations of the nonthyroidal illness syndrome in this model; 2) the lack of D3 does not affect the extent of pulmonary inflammation; and 3) bacterial outgrowth in blood, spleen, and lung of D3KO mice is significantly higher than in WT mice. Our results suggest a protective role for D3 in the defense against acute bacterial infection, probably by reinforcing the microbial killing capacity of neutrophils.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M.bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M.marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M.bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M.bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd.

Background CD14 is a pattern-recognition receptor that can facilitate the presentation of bacterial components to either Toll-like receptor 2 (TLR2) or TLR4. We have recently shown that during melioidosis, a severe infection caused by the gram-negative bacterium Burkholderia pseudomallei, TLR2 but not TLR4 impacts the immune response of the intact host in vivo. Methods The function of CD14 in melioidosis was analyzed by means of in vitro and in vivo approaches, using wild-type (WT) and CD14 knockout (KO) mice. Results CD14-deficient macrophages and whole blood leukocytes released less tumor necrosis factor (TNF)-alpha on stimulation with B. pseudomallei or B. pseudomallei lipopolysaccharide in vitro, compared with WT cells. Strikingly, CD14 KO mice intranasally inoculated with B. pseudomallei demonstrated reduced lethality and significantly decreased bacterial outgrowth, compared with WT mice. Administration of recombinant soluble CD14 to CD14 KO mice partially reversed their phenotype to that of WT mice. Lastly, CD14 deficiency did not alter the capacity of macrophages or neutrophils to phagocytose or kill B. pseudomallei. Conclusion CD14 is crucially involved in the recognition of B. pseudomallei by innate immune cells but plays a remarkable detrimental role in the host response against B. pseudomallei. Inhibition of CD14 may be a novel treatment strategy in melioidosis.

Nontypeable Haemophilus influenzae (NTHi) is a common gram-negative respiratory pathogen. To determine the role of the proinflammatory cytokine interleukin 18 (IL-18) during NTHi pneumonia, normal wild-type (WT) and IL-18 knockout (KO) mice were intranasally infected with NTHi. IL-18 KO mice displayed a delayed clearance of NTHi from the respiratory tract, resulting in >20-fold higher bacterial loads in their lungs at 24 h after infection, preceded by a strongly attenuated pulmonary innate immune response as determined by cytokine and chemokine induction and histopathology. These data identify IL-18 as part of an adequate innate immune response during NTHi pneumonia.

Mycobacterium kansasii is an emerging pathogen that is able to induce pulmonary disease resembling tuberculosis. To determine the role of interleukin (IL-)1 in lung infection caused by this atypical mycobacterium, IL-1 receptor type 1 knockout (IL-1R(1) KO) and normal wild type mice were intranasally infected with M. kansasii. IL-1R(1) KO mice demonstrated a reduced antibacterial response in the lungs and an increased dissemination to the liver, which was accompanied by an enhanced pulmonary inflammatory response. These data identify IL-1 as an important component of the innate immune response to lung infection by M. kansasii.

Mycobacterium kansasii has emerged as an important nontuberculous mycobacterium that can cause severe infection in the immunocompromised host, especially in human immunodeficiency virus-infected patients. However, little is known about the pathogenesis of this infection. Because patients suffering from M. kansasii infection are severely compromised in their cellular immune response, we studied the course of infection in CD4+ cell knockout (KO) mice. Wild-type (WT) mice and CD4+ KO mice were infected with 10(5) cfu of M. kansasii. Although previously shown to be susceptible to Mycobacterium tuberculosis infection, CD4+ KO mice demonstrated no impairment in clearing infection with M. kansasii when compared with WT animals, despite reduced pulmonary inflammation (reduced granuloma formation and lymphocyte infiltration in the lungs). Pulmonary IFN-gamma levels and M. kansasii-induced IFN-gamma production by splenocytes from infected animals were reduced in CD4+ KO mice, confirming that these mice were defective in the M. kansasii-specific T helper cell type 1 immune response. Furthermore, mice deficient for IFN-gamma, IL-12p35, IL-12p40, or IL-18 also displayed a normal host defense against pulmonary infection with M. kansasii. These data suggest that CD4+ cells, IFN-gamma, and an intact T helper cell type 1 response play a limited role in protective immunity against pulmonary M. kansasii infection.

The development of a strong T(h)1-mediated adaptive immune response is considered of main importance for host defense against the intracellular pathogen Mycobacterium tuberculosis. The induction of a cellular immune response is not only dependent on the engagement of the TCR but also requires co-stimulation. In order to study the role of the co-stimulatory molecule of the tumor necrosis factor receptor family member CD27 during murine M. tuberculosis infection, we intranasally infected wild-type (WT) and CD27 knockout (KO) mice with 10(5) colony-forming units M. tuberculosis. Whereas there were no differences in bacterial growth, inflammation and IFN gamma production by CD4(+) and CD8(+) lymphocytes in the lungs early after infection, the number of splenic CD8(+) T cells producing the key T(h)1 cytokine IFN gamma was lower in CD27 KO mice than in WT mice. After 6 weeks, CD27 KO mice had 3.6-fold higher mycobacterial counts in their lungs and displayed more pulmonary inflammation and increased numbers of infiltrated leukocytes. Despite these differences early in infection, an equal number of WT and CD27 KO mice died during a 43-week observation period and lung bacterial loads and inflammation were comparable in the surviving animals. Our data suggest that CD27 does not contribute to the local IFN gamma-mediated response and long-term protection against M. tuberculosis.

RATIONALE: Acinetobacter baumannii is an opportunistic bacterial pathogen that is increasingly associated with gram-negative nosocomial pneumonia, but the molecular mechanisms that play a role in innate defenses during A. baumannii infection have not been elucidated. OBJECTIVE: To gain first insight into the role of CD14 and Toll-like receptors 4 and 2 in host response to A. baumannii pneumonia. METHODS: Respective gene-deficient mice were intranasally infected with A. baumannii, and bacterial outgrowth, lung inflammation, and pulmonary cytokine/chemokine responses were determined. To study the importance of LPS in the inflammatory response, mice were also challenged with A. baumannii LPS. MEASUREMENTS AND MAIN RESULTS: Bacterial counts were increased in CD14 and Toll-like receptor 4 gene-deficient mice, and only these animals developed bacteremia. The pulmonary cytokine/chemokine response was impaired in Toll-like receptor 4 knockout mice and the onset of lung inflammation was delayed. In contrast, Toll-like receptor 2-deficient animals displayed an earlier cell influx into lungs combined with increased macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 concentrations, which was associated with accelerated elimination of bacteria from the pulmonary compartment. Neither CD14 nor Toll-like receptor 4 gene-deficient mice responded to intranasal administration of LPS, whereas Toll-like receptor 2 knockout mice were indistinguishable from wild-type animals. CONCLUSIONS: Our results suggest that CD14 and Toll-like receptor 4 play a key role in innate sensing of A. baumannii via the LPS moiety, resulting in effective elimination of the bacteria from the lung, whereas Toll-like receptor 2 signaling seems to counteract the robustness of innate responses during acute A. baumannii pneumonia

Interleukin (IL)-12 (p70) is a heterodimeric cytokine composed of p40 and p35, that plays a major role in the protective immune response to Mycobacterium tuberculosis. To define the role of p40 in lungs during pulmonary M. tuberculosis infection we generated transgenic (Tg) mice overexpressing p40 under control of the surfactant protein C promoter. Tg mice expressed the transgene in their lungs, yet demonstrated elevated pulmonary p40 protein levels. After infection, Tg mice displayed higher pulmonary p40 and p70 levels than wild type mice. Interferon-gamma concentrations were similar in uninfected and infected Tg and wild type mice, arguing against agonistic effects of p40. Tg mice demonstrated reduced recruitment of macrophages, lymphocytes and neutrophils to the lungs early after infection. This was accompanied by reduced pulmonary tumour necrosis factor-alpha, macrophage inflammatory protein (MIP)-2 and MIP-1alpha levels. This suggests that elevated p40 concentrations inhibited the chemotactic effects of p70 on leucocytes. Furthermore, Tg mice displayed slightly higher pulmonary mycobacterial outgrowth late in the infection than wild type mice. Taken together, we demonstrate that constitutive overexpression of p40 in lungs negatively influences IL-12-mediated leucocyte migration and protection against lung tuberculosis. This suggests a novel antagonistic role for p40 homodimers in regulating the chemotactic bioactivity of IL-12 after pulmonary mycobacterial infection.

Leptin is a pleiotrophic protein mainly produced by adipocytes that has been implicated as a link between nutritional status and immune function. Severe bacterial infection is associated with elevated plasma levels of leptin. To determine the role of leptin in the host response to bacterial pneumonia leptin deficient ob/ob mice and normal wild-type (WT) mice were intranasally infected with different doses of the Gram-positive pathogen Streptococcus (S.) pneumoniae or the Gram-negative bacterium Klebsiella (K.) pneumoniae. After infection with lower doses of either pathogen ob/ob mice displayed lower pulmonary levels of proinflammatory cytokines, in particular tumor necrosis factor-alpha and chemokines. However, after infection with a higher dose of S. pneumoniae or K. pneumoniae the lung concentrations of these inflammatory mediators did not differ between ob/ob and WT mice. In addition, the extent and severity of lung inflammation, as assessed by semi-quantitative histopathology scores, were similar in both mouse strains. Finally, leptin deficiency did not impact on the bacterial outgrowth in the lungs during either Gram-positive or Gram-negative pneumonia irrespective of the infective dose. These data suggest that although leptin may play a modest role in the regulation of inflammation during bacterial pneumonia, it does not contribute to host defense mechanisms that act to limit the outgrowth of S. pneumoniae or K. pneumoniae in the lower airways.

Toll-like receptors (TLR) are crucial pattern recognition receptors in innate immunity. The importance of TLR2 in host defense against Gram-positive bacteria has been suggested by the fact that this receptor recognizes major Gram-positive cell wall components, such as peptidoglycan and lipoteichoic acid. To determine the role of TLR2 in pulmonary Gram-positive infection, we first established that TLR2 is indispensable for alveolar macrophage responsiveness toward Streptococcus pneumoniae. Nonetheless, TLR2 gene-deficient mice intranasally inoculated with S. pneumoniae at doses varying from nonlethal (with complete clearance of the infection) to lethal displayed only a modestly reduced inflammatory response in their lungs and an unaltered antibacterial defense when compared with normal wild-type mice. These data suggest that TLR2 plays a limited role in the innate immune response to pneumococcal pneumonia, and that additional pattern recognition receptors likely are involved in host defense against this common respiratory pathogen.

Chronic pulmonary infection withPseudomonas aeruginosais common in cystic fibrosis (CF) patients.P. aeruginosalipopolysaccharide (LPS), phosholipase C (PLC), and exotoxin A (ETA) were evaluated for their ability to induce pulmonary inflammation in mice following intranasal inoculation. Both LPS and PLC induced high levels of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), IL-6, gamma interferon (IFN-γ), MIP-1α and MIP-2 in the lungs but did not affect IL-18 levels. ETA did not induce TNF-α and was a weak inducer of IL-1β, IL-6, macrophage inflammatory protein 1α (MIP-1α), and MIP-2. Remarkably, ETA reduced constitutive lung IL-18 levels. LPS was the only factor inducing IFN-γ. LPS, PLC, and ETA all induced cell infiltration in the lungs. The role of interferon regulatory factor-1 (IRF-1) in pulmonary inflammation induced by LPS, PLC, and ETA was evaluated. When inoculated with LPS, IRF-1 gene knockout (IRF-1 KO) mice produced lower levels of TNF-α, IL-1β, and IFN-γ than did wild-type (WT) mice. Similarly, a milder effect of ETA on IL-1β and IL-18 was observed for IRF-1 KO than for WT mice. In contrast, the cytokine response to PLC did not differ between WT and IRF-1 KO mice. Accordingly, LPS and ETA, but not PLC, induced expression of IRF-1 mRNA. IRF-1 deficiency had no effect on MIP-1α and MIP-2 levels and on cell infiltration induced by LPS, PLC, or ETA. Flow cytometric evaluation of lung mononuclear cells revealed strongly reduced percentages of CD8+and NK cells in IRF-1 KO mice compared to percentages observed for WT mice. These data indicate that different virulence factors fromP. aeruginosainduce pulmonary inflammation in vivo and that IRF-1 is involved in some of the cytokine responses to LPS and ETA.

Background Mechanical ventilation (MV) can cause ventilator-induced lung injury (VILI). The innate immune response mediates this iatrogenic inflammatory condition. The receptor for advanced glycation end products (RAGE) is a multiligand receptor that can amplify immune and inflammatory responses. We hypothesized that RAGE signaling contributes to the pro-inflammatory state induced by MV. Methods RAGE expression was analyzed in lung brush and lavage cells obtained from ventilated patients and lung tissue of ventilated mice. Healthy wild-type (WT) and RAGE knockout (KO) mice were ventilated with relatively low (approximately 7.5 ml/kg) or high (approximately 15 ml/kg) tidal volume. Positive end-expiratory pressure was set at 2 cm H2O during both MV strategies. Also, WT and RAGE KO mice with lipopolysaccharide (LPS)-induced lung injury were ventilated with the above described ventilation strategies. In separate experiments, the contribution of soluble RAGE, a RAGE isoform that may function as a decoy receptor, in ventilated RAGE KO mice was investigated. Lung wet-to-dry ratio, cell and neutrophil influx, cytokine and chemokine concentrations, total protein levels, soluble RAGE, and high-mobility group box 1 (HMGB1) presence in lung lavage fluid were analyzed. Results MV was associated with increased RAGE mRNA levels in both human lung brush samples and lung tissue of healthy mice. In healthy high tidal volume-ventilated mice, RAGE deficiency limited inflammatory cell influx. Other VILI parameters were not affected. In our second set of experiments where we compared RAGE KO and WT mice in a 2-hit model, we observed higher pulmonary cytokine and chemokine levels in RAGE KO mice undergoing LPS/high tidal volume MV as compared to WT mice. Third, in WT mice undergoing the LPS/high tidal volume MV, we observed HMGB1 presence in lung lavage fluid. Moreover, MV increased levels of soluble RAGE in lung lavage fluid, with the highest levels found in LPS/high tidal volume-ventilated mice. Administration of soluble RAGE to LPS/high tidal volume-ventilated RAGE KO mice attenuated the production of inflammatory mediators. Conclusions RAGE was not a crucial contributor to the pro-inflammatory state induced by MV. However, the presence of sRAGE limited the production of pro-inflammatory mediators in our 2-hit model of LPS and high tidal volume MV. Electronic supplementary material The online version of this article (doi:10.1186/s40635-014-0022-1) contains supplementary material, which is available to authorized users.

Innate immunity pathways are found to play an important role in ventilator-induced lung injury. We analyzed pulmonary expression of Toll-like receptor 2 (TLR2) in humans and mice and determined the role of TLR2 in the pathogenesis of ventilator-induced lung injury in mice. Toll-like receptor 2 gene expression was analyzed in human bronchoalveolar lavage fluid (BALF) cells and murine lung tissue after 5 h of ventilation. In addition, wild-type (WT) and TLR2 knockout (KO) mice were ventilated with either lower tidal volumes (VT) of 7 mL/kg with positive end-expiratory pressure (PEEP) or higher VT of 15 mL/kg without PEEP for 5 h. Spontaneously breathing mice served as controls. Total protein and immunoglobulin M levels in BALF, neutrophil influx into the alveolar compartment, and interleukin 6 (IL-6), IL-1β, and keratinocyte-derived chemokine concentrations in lung tissue homogenates were measured. We observed enhanced TLR2 gene expression in BALF cells of ventilated patients and in lung tissue of ventilated mice. In WT mice, ventilation with higher VT without PEEP resulted in lung injury and inflammation with higher immunoglobulin M levels, neutrophil influx, and levels of inflammatory mediators compared with controls. In TLR2 KO mice, neutrophil influx and IL-6, IL-1β, and keratinocyte-derived chemokine were enhanced by this ventilation strategy. Ventilation with lower VT with PEEP only increased neutrophil influx and was similar in WT and TLR2 KO mice. In summary, injurious ventilation enhances TLR2 expression in lungs. Toll-like receptor 2 deficiency does not protect lungs from ventilator-induced lung injury. In contrast, ventilation with higher VT without PEEP aggravates inflammation in TLR2 KO mice.

Adenosine monophosphate-activated protein (AMP)-activated kinase (AMPK) is a highly conserved kinase that plays a key role in energy homeostasis. Activation of AMPK was shown to reduce inflammation in response to lipolysaccharide in vitro and in vivo. 5-Aminoimidazole-4-carbox-amide-1-β-d-ribofuranoside (AICAR) is intracellularly converted to the AMP analog ZMP, which activates AMPK. Lipoteichoic acid (LTA) is a major component of the cell wall of Gram-positive bacteria that can trigger inflammatory responses. In contrast to lipopolysaccharide, little is known on the effects of AMPK activation in LTA-triggered innate immune responses. Here, we studied the potency of AMPK activation to reduce LTA-induced inflammation in vitro and in lungs in vivo. Activation of AMPK in vitro reduced cytokine production in the alveolar macrophage cell line MH-S. In vivo, AMPK activation reduced LTA-induced neutrophil influx, as well as protein leak and cytokine/chemokine levels in the bronchoalveolar space. In conclusion, AMPK activation inhibits LTA-induced lung inflammation in mice.

Background Gram-positive and gram-negative bacteria are common causative agents of respiratory tract infection. Lipopolysaccharide (LPS) is a component of the gram-negative cell wall and a strong inducer of inflammation. The main proinflammatory component of the gram-positive bacterial cell wall is lipoteichoic acid (LTA). The protein kinase p38 mitogen activated protein kinase (MAPK) plays an important role in the inflammatory process induced by these two bacterial structures. Aim We here sought to establish the impact of local p38 MAPK inhibition on lung inflammatory responses induced by LPS and LTA. We investigated the effects of direct intrapulmonary delivery of a p38 MAPK inhibitor on local LPS and LTA induced airway inflammation in mice. Results In vitro , BIRB 796 reduced LPS induced p38 MAPK phosphorylation in alveolar macrophage and respiratory epithelial cell lines and diminished cytokine/chemokine release. In vivo , BIRB 796 circumvented p38 MAPK phosphorylation in both LPS and LTA induced inflammation. Cellular influx was not affected. Lung TNFα, IL-6, MIP-2 and LIX production was reduced in LPS induced inflammation but not in lung inflammation by LTA. BIRB 796 reduced total protein and IgM in bronchoalveolar lavage fluid after LTA instillation, while enhancing TATc and d -dimers in LPS- and LTA induced inflammation. Conclusion These results taken together with earlier studies on systemic administration of p38 MAPK inhibitors in rodents and humans suggest that direct intrapulmonary delivery of a p38 MAPK inhibitor is less effective in inhibiting inflammation and is associated with unexpected procoagulant effects in the bronchoalveolar space.

BACKGROUND: Bacterial products add to mechanical ventilation in enhancing lung injury. The role of endogenous triggers of innate immunity herein is less well understood. S100A8/A9 proteins are released by phagocytes during inflammation. The present study investigates the role of S100A8/A9 proteins in ventilator-induced lung injury. METHODS: Pulmonary S100A8/A9 levels were measured in samples obtained from patients with and without lung injury. Furthermore, wild-type and S100A9 knock-out mice, naive and with lipopolysaccharide-induced injured lungs, were randomized to 5 hours of spontaneously breathing or mechanical ventilation with low or high tidal volume (VT). In addition, healthy spontaneously breathing and high VT ventilated mice received S100A8/A9, S100A8 or vehicle intratracheal. Furthermore, the role of Toll-like receptor 4 herein was investigated. RESULTS: S100A8/A9 protein levels were elevated in patients and mice with lung injury. S100A8/A9 levels synergistically increased upon the lipopolysaccharide/high VT MV double hit. Markers of alveolar barrier dysfunction, cytokine and chemokine levels, and histology scores were attenuated in S100A9 knockout mice undergoing the double-hit. Exogenous S100A8/A9 and S100A8 induced neutrophil influx in spontaneously breathing mice. In ventilated mice, these proteins clearly amplified inflammation: neutrophil influx, cytokine, and chemokine levels were increased compared to ventilated vehicle-treated mice. In contrast, administration of S100A8/A9 to ventilated Toll-like receptor 4 mutant mice did not augment inflammation. CONCLUSION: S100A8/A9 proteins increase during lung injury and contribute to inflammation induced by HVT MV combined with lipopolysaccharide. In the absence of lipopolysaccharide, high levels of extracellular S100A8/A9 still amplify ventilator-induced lung injury via Toll-like receptor 4.

SummaryThe protein C (PC) system is an important regulator of both coagulation and inflammation. Activated PC (APC), together with its receptor the endothelial protein C receptor (EPCR), has anticoagulant and anti-inflammatory properties. During tuberculosis (TB), a devastating chronic pulmonary disease caused by Mycobacterium (M.) tuberculosis, both a local inflammatory reaction characterised by the recruitment of mainly mononuclear cells and the formation of pulmonary granulomas as well as activation of coagulation occurs as part of the host immune response. We investigated the role of EPCR and APC in a mouse model of TB using mice overexpressing EPCR (Tie2-EPCR), mice deficient for EPCR (EPCR−/−), mice treated with APC-inhibiting antibodies and mice overexpressing APC (APChigh) and compared them with wild-type (WT) mice. Blood and organs were harvested to quantify bacterial loads, cellular influxes, cytokines, histopathology and coagulation parameters. Additionally observation studies were performed. Lung EPCR expression was upregulated during experimental TB. No significant differences in bacterial growth were seen between WT and Tie2-EPCR mice. However, Tie2-EPCR mice had decreased pulmonary coagulation activation, displayed an increased influx of macro-phages 2 and 6 weeks after infection, but no increase in other proin-flammatory markers. On the other hand, in EPCR−/−-mice coagulation activation was decreased 6 weeks post-infection, with little impact on other inflammation markers. APC-overexpression or treatment with anti-(A)PC antibodies displayed minimal effects during experimental TB. In conclusion, EPCR and APC play a limited role in the host response during experimental pulmonary TB.

Introduction Uric acid released from injured tissue is considered a major endogenous danger signal and local instillation of uric acid crystals induces acute lung inflammation via activation of the NLRP3 inflammasome. Ventilator-induced lung injury (VILI) is mediated by the NLRP3 inflammasome and increased uric acid levels in lung lavage fluid are reported. We studied levels in human lung injury and the contribution of uric acid in experimental VILI. Methods Uric acid levels in lung lavage fluid of patients with acute lung injury (ALI) were determined. In a different cohort of cardiac surgery patients, uric acid levels were correlated with pulmonary leakage index. In a mouse model of VILI the effect of allopurinol (inhibits uric acid synthesis) and uricase (degrades uric acid) pre-treatment on neutrophil influx, up-regulation of adhesion molecules, pulmonary and systemic cytokine levels, lung pathology, and regulation of receptors involved in the recognition of uric acid was studied. In addition, total protein and immunoglobulin M in lung lavage fluid and pulmonary wet/dry ratios were measured as markers of alveolar barrier dysfunction. Results Uric acid levels increased in ALI patients. In cardiac surgery patients, elevated levels correlated significantly with the pulmonary leakage index. Allopurinol or uricase treatment did not reduce ventilator-induced inflammation, IκB-α degradation, or up-regulation of NLRP3, Toll-like receptor 2, and Toll-like receptor 4 gene expression in mice. Alveolar barrier dysfunction was attenuated which was most pronounced in mice pre-treated with allopurinol: both treatment strategies reduced wet/dry ratio, allopurinol also lowered total protein and immunoglobulin M levels. Conclusions Local uric acid levels increase in patients with ALI. In mice, allopurinol and uricase attenuate ventilator-induced alveolar barrier dysfunction.

Background Helium inhalation protects myocardium, brain and endothelium against ischemia/reperfusion injury in animals and humans, when applied according to specific “conditioning” protocols. Before widespread use of this “conditioning” agent in clinical practice, negative side effects have to be ruled out. We investigated the effect of prolonged helium inhalation on the responsiveness of the human immune response in whole blood ex vivo. Methods Male healthy volunteers inhaled 30 minutes heliox (79%He/21%O2) or air in a cross over design, with two weeks between measurements. Blood was withdrawn at T0 (baseline), T1 (25 min inhalation) and T2-T5 (1, 2, 6, 24 h after inhalation) and incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), T-cell stimuli anti-CD3/ anti-CD28 (TCS) or RPMI (as control) for 2, 4 and 24 hours or not incubated (0 h). An additional group of six volunteers inhaled 60 minutes of heliox or air, followed by blood incubation with LPS and RPMI. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ) and interleukin-2 (IL-2) was analyzed by cytometric bead array. Statistical analysis was performed by the Wilcoxon test for matched samples. Results Incubation with LPS, LTA or TCS significantly increased TNF-α, IL-1β, IL-6, IL-8, IFN-γ and IL-2 in comparison to incubation with RPMI alone. Thirty min of helium inhalation did not influence the amounts of TNF-α, IL-1β, IL-6, IL-8, IFN-γ and IL-2 in comparison to air. Sixty min of helium inhalation did not affect cytokine production after LPS stimulation. Conclusions We conclude that 79% helium inhalation does not affect the responsiveness of the human immune system in healthy volunteers. Trial registration Dutch Trial Register:http://www.trialregister.nl/ NTR2152

Melioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei , is an important cause of community-acquired sepsis in Southeast Asia and northern Australia. An important controller of the immune system is the pleiotropic cytokine transforming growth factor β (TGF-β), of which Smad2 and Smad3 are the major signal transducers. In this study, we aimed to characterize TGF-β expression and function in experimental melioidosis. TGF-β expression was determined in 33 patients with culture-proven infection with B. pseudomallei and 30 healthy controls. We found that plasma TGF-β concentrations were strongly elevated during melioidosis. In line with this finding, TGF-β expression in C57BL/6 mice intranasally inoculated with B. pseudomallei was enhanced as well. To assess the role of TGF-β, we inhibited TGF-β using a selective murine TGF-β antibody. Treatment of mice with anti-TGF-β antibody resulted in decreased lung Smad2 phosphorylation. TGF-β blockade appeared to be protective: mice treated with anti-TGF-β antibody and subsequently infected with B. pseudomallei showed diminished bacterial loads. Moreover, less distant organ injury was observed in anti-TGF-β treated mice as shown by reduced blood urea nitrogen (BUN) and aspartate transaminase (AST) values. However, anti-TGF-β treatment did not have an effect on survival. In conclusion, TGF-β is upregulated during B. pseudomallei infection and plays a limited but proinflammatory role during experimental melioidosis.

Mechanical ventilation (MV) has the potential to worsen pre-existing lung injury or even to initiate lung injury. Moreover, it is thought that injurious MV contributes to the overwhelming inflammatory response seen in patients with acute lung injury or acute respiratory distress syndrome. Ventilator-induced lung injury (VILI) is characterized by increased endothelial and epithelial permeability and pulmonary inflammation, in which the innate immune system plays a key role. A growing body of evidence indicates that endogenous danger molecules, also termed damage-associated molecular patterns (DAMPs), are released upon tissue injury and modulate the inflammatory response. DAMPs activate pattern recognition receptors, may induce the release of proinflammatory cytokines and chemokines, and have been shown to initiate or propagate inflammation in non-infectious conditions. Experimental and clinical studies demonstrate the presence of DAMPs in bronchoalveolar lavage fluid in patients with VILI and the upregulation of pattern recognition receptors in lung tissue by MV. The objective of the present article is to review research in the area of DAMPs, their recognition by the innate immune system, their role in VILI, and the potential utility of blocking DAMP signaling pathways to reduce VILI in the critically ill.

The development of active tuberculosis after infection with Mycobacterium tuberculosis is almost invariably associated with a persistent or transient state of relative immunodeficiency. The receptor for advanced glycation end products (RAGE) is a promiscuous receptor that is involved in pulmonary inflammation and infection. To investigate the role of RAGE in tuberculosis, we intranasally infected wild-type (Wt) and RAGE deficient (RAGE(-/-)) mice with live virulent M. tuberculosis. While lungs of uninfected Wt mice expressed RAGE, in particular on endothelium, M. tuberculosis pneumonia was associated with an enhanced pulmonary expression of RAGE. Lung inflammation was increased in RAGE(-/-) mice, as indicated by histopathology, percentage of inflamed area, lung weight and cytokine and chemokine levels. In addition, lung lymphocyte and neutrophil numbers were increased in the RAGE(-/-) mice. RAGE(-/-) mice had modestly higher mycobacterial loads in the lungs after 3 weeks but not after 6 weeks of infection. Moreover, RAGE(-/-) mice displayed more body weight loss and enhanced mortality. In summary, pulmonary RAGE expression is increased during tuberculosis. In addition, these data suggest that RAGE plays a beneficial role in the host response to pulmonary tuberculosis. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved

Background. Pneumonia is frequently caused by gram-negative pathogens, among which Klebsiella pneumoniae prominently features. Recognition of pathogen-associated molecular patterns by Toll-like receptors (TLRs) is important for an appropriate immune response during infection. TLR signaling can proceed via 2 distinct routes that are dependent on the adaptor proteins Myeloid differentiation primary response gene (88) (MyD88) and TIR-domain-containing adaptor-inducing interferon-beta (TRIF). The aim of the study was to determine the relative contribution of MyD88 and TRIF signaling in resident and hematopoietic cells to host defense during pneumonia. Methods. Bone marrow chimeras of MyD88 deficient/wild type and TRIF mutant/wild type mice were created and infected with K. pneumoniae via the airways. Results. MyD88 in both resident and hematopoietic cells contributed to survival and antibacterial defense in late-stage infection, whereas only TRIF in hematopoietic cells was protective. On the other hand, resident MyD88 and hematopoietic TRIF contributed to distant cellular injury. Resident MyD88 was pivotal for early chemokine release and neutrophil recruitment in the bronchoalveolar space. Conclusions. MyD88- and TRIF-dependent signaling has a differential contribution to host defense in different cell types that changes from early-to late-stage gram-negative pneumonia

Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths worldwide. Non-specific host defense mechanisms such as the coagulation and fibrinolytic system may give insight in possible new therapeutic targets. Plasminogen activator inhibitor type-1 (PAI-1), an important regulator of inflammation and fibrinolysis, might be of interest as tuberculosis patients have elevated plasma levels of PAI-1. In this study we set out to investigate the role of PAI-1 during tuberculosis in vivo. Wildtype (WT) and PAI-1 deficient (PAI-1(-/-)) mice were intranasally infected with M. tuberculosis H37rv and sacrificed after 2,5 and 29 weeks. Five weeks post-infection, bacterial loads in lungs of PAI-1(-/-) mice were significantly higher compared to WT mice, while no differences were seen 2 and 29 weeks post-infection. At two weeks post-infection increased influx of macrophages and lymphocytes was observed. PAI-1 deficiency was associated with a reduced cytokine response in the lungs; however, upon stimulation with tuberculin purified protein derivative (PPD), PAI-1(-/-) splenocytes released increased levels of IFN-gamma compared to WT. No clear differences were found between PAI-1(-/-) and WT mice at 29 weeks after infection. In conclusion, these data suggest that PAI-1 contributes to transient, non-specific changes in immunity during the early phase of murine tuberculosis. Crown Copyright (C) 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur. All rights reserved

In this study, the relative roles of Toll-like receptor (TLR)2 and TLR4 were investigated independently and together. Moreover, we studied the role of haematopoietic compartment in anti-Klebsiella host defence. We infected TLR2 and TLR4 single-, and TLR2×4 double knockout (KO) animals with different doses of Klebsiella pneumoniae. In addition, bone marrow chimeric mice were created and infected. TLR4 played a more prominent role in antibacterial defence than TLR2, considering that only TLR4 KO mice demonstrated enhanced bacterial growth in lungs and spleen 24 h after infection with 3×10³ colony-forming units of Klebsiella compared with wild-type (WT) mice. In late-stage infection or after exposure to a higher infectious dose, bacterial counts in lungs of TLR2 KO animals were elevated compared with WT mice and TLR2×4 KO animals were more susceptible to infection than TLR4 KO mice. TLR signalling in cells of haematopoietic origin is of primary importance in host defence against K. pneumoniae. These data suggest that: 1) TLR4 drives the antibacterial host response after induction of pneumonia with relatively low Klebsiella doses; 2) TLR2 becomes involved at a later phase of the infection and/or upon exposure to higher bacterial burdens; and 3) haematopoietic TLR2 and TLR4 are important for an adequate host response during Klebsiella pneumonia.

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 108 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-a 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 WTCD4(+) and CD8(+) T cells to produce IFN-gamma and tumor necrosis factor-a, 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

Non-typeable Haemophilus influenzae (NTHi) is a common Gram-negative respiratory pathogen. We demonstrated previously that myeloid differentiation primary-response protein 88 (MyD88) is of utmost importance in host defense against NTHi. All TLRs except for TLR3 depend on MyD88 for signaling. TLR9, the TLR for detecting pathogen DNA depends on MyD88 signaling. Here, we investigate the role of TLR9 during NTHi pneumonia. Alveolar macrophages (AM) from normal wild-type (WT) and TLR9 knock-out (KO) mice were harvested and stimulated with growth-arrested NTHi or CPG DNA. WT and TLR9 KO mice were infected intranasally with NTHi: cytokine and chemokine responses were measured 16h later. Despite significant reduced TNF production by TLR9 KO AM in response to CPG DNA, no difference was detected in TNF production after NTHi stimulation by isolated alveolar macrophages from WT and TLR9 KO mice. Moreover, we found similar pulmonary bacterial loads, similar cytokine and chemokine levels in WT and TLR9 KO mice, and no differences in histopathology. In conclusion, we were not able to demonstrate a role for TLR9 in the recognition of and host defense against NTHi.

BACKGROUND: S-ketamine is frequently used for analgosedation, especially during sepsis and cardiovascular instability. Because S-ketamine blocks voltage-gated sodium (Na+) channels in neurons and skeletal muscle, it is conceivable that S-ketamine also blocks alveolar epithelial Na+ channels that are crucial for alveolar fluid clearance (AFC). We studied the effects of alveolar and IV S-ketamine on transalveolar Na+ transport and AFC, and investigated whether IV S-ketamine enters the alveolar space in response to endotoxemia-induced pulmonary inflammation. METHODS: Cultured rat alveolar type II (ATII) cells were exposed to S-ketamine and/or the Na+ channel blocker amiloride (100 microM) and transepithelial transport indicated by short circuit current (ISC) was measured in Ussing chambers. AFC was measured in fluid-instilled lungs of anesthetized rats with or without amiloride added to the instillate. S-ketamine was either added to the instillate or injected IV. To induce mild lung injury that might favor the appearance of IV S-ketamine at the alveolar surface, endotoxemia was induced by IV lipopolysaccharide (7.5 mg/kg). RESULTS: In ATII cells, S-ketamine (25 microg/mL) caused a decrease of ISC regardless of apical (-18.9%+/- 1.4%; P

Upon infection with Mycobacterium (M.) tuberculosis, the development of a strong T helper 1 (Th1)-mediated adaptive immune response is considered as being most important for containment of the infection. Osteopontin (OPN) is a phosphorylated glycoprotein that is chemotactic for inflammatory cells and has been implicated in the induction of Th1 responses and granulomatous disease. We tested the hypothesis that OPN facilitates protective immunity during M. tuberculosis infection using wild-type (WT) and OPN knockout (KO) mice in a model of pulmonary tuberculosis. OPN expression was up-regulated in alveolar macrophages and lymphoid cells during M. tuberculosis infection. There were no significant differences in bacterial outgrowth, inflammation or recruitment of lymphocytes, macrophages and polymorphonuclear cells in the lungs after 2 and 5 weeks of infection. However, the numbers of CD4(+) and CD8(+) T cells were reduced in the absence of OPN 5 weeks after infection. Similar concentrations of cytokine were observed in lungs from both WT mice and OPN KO mice; however, there was a trend towards decreased levels of interferon-gamma (IFN-gamma) in OPN KO mice 5 weeks after infection. Despite an unaltered immune response in the early phase of tuberculosis, OPN KO mice had a modest survival advantage. Of note, both pulmonary bacterial loads and lung inflammation were reduced in these mice 31 weeks after infection. These data suggest that OPN is not crucial for protective immunity upon M. tuberculosis infection and during the late phase of tuberculosis may even be detrimental for the host.

Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths world-wide every year. Successful host defense mainly depends on a strong Th type I response. We investigated the role of T1/ST2 (recently identified as the receptor for IL-33), a typical Th2 marker in the assumption that a shift towards a beneficial Th I response would Occur in the absence of ST2. For this, ST2 KO and WT mice were intranasally infected with a virulent strain of M. tuberculosis (150 CFU). In line with our hypothesis, ST2 KO animals displayed increased numbers of lymphocytes infiltrating the lung after 2 weeks of infection, increased IFN gamma production by splenocytes in ST2 KO mice early in infection and enhanced lung IFN gamma levels at the chronic phase of the disease. However, we did not detect any differences between ST2 KO and WT mice in mycobacterial loads in lungs or liver after M. tuberculosis infection. The pulmonary inflammatory response, as measured by relative lung weights, cytokine and chemokine levels as well as histopathological analysis, was similar in ST2 KO and WT mice. These data suggest that apart from inducing a modest shift towards the Th I response, the role of ST2 during murine M. tuberculosis infection is limited. (C) 2009 Elsevier Masson SAS. All rights reserved

Toll-like receptors play an essential role in the innate recognition of micro-organisms by the host. CD14 is one of the extracellular adaptor proteins required for recognition of Gram-negative bacteria and possibly also Mycobacterium tuberculosis. Therefore, we intranasally infected wild-type (WT) and CD14 knock-out (KO) mice with virulent M. tuberculosis H37Rv. We found no differences in bacterial load in the main target organ lung up to 32 weeks after infection. From 20 weeks onward 57% of WT mice succumbed, whereas all CD14 KO mice survived. The improved outcome of CD14 KO mice was accompanied by reduced pulmonary inflammation; lung cell counts and percentage of inflamed lung tissue were reduced in CD14 WT mice. These data suggest that during chronic infection CD14 KO mice are protected from lethality caused by lung tuberculosis because of a reduction of the inflammatory response.

Background: Toll-like receptors (TLRs) have a central role in the recognition of pathogens and the initiation of the innate immune response. Myeloid differentiation primary-response gene 88 (MyD88) and TIR-domain-containing adaptor protein inducing IFN beta (TRIF) are regarded as the key signaling adaptor proteins for TLRs. Melioidosis, which is endemic in SE-Asia, is a severe infection caused by the gram-negative bacterium Burkholderia pseudomallei. We here aimed to characterize the role of MyD88 and TRIF in host defense against melioidosis. Methodology and Principal Findings: First, we found that MyD88, but not TRIF, deficient whole blood leukocytes released less TNF alpha upon stimulation with B. pseudomallei compared to wild-type (WT) cells. Thereafter we inoculated MyD88 knockout (KO), TRIF mutant and WT mice intranasally with B. pseudomallei and found that MyD88 KO, but not TRIF mutant mice demonstrated a strongly accelerated lethality, which was accompanied by significantly increased bacterial loads in lungs, liver and blood, and grossly enhanced liver damage compared to WT mice. The decreased bacterial clearance capacity of MyD88 KO mice was accompanied by a markedly reduced early pulmonary neutrophil recruitment and a diminished activation of neutrophils after infection with B. pseudomallei. MyD88 KO leukocytes displayed an unaltered capacity to phagocytose and kill B. pseudomallei in vitro. Conclusions: MyD88 dependent signaling, but not TRIF dependent signaling, contributes to a protective host response against B. pseudomallei at least in part by causing early neutrophil recruitment towards the primary site of infection

Background Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis. Methods and Findings Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury. Conclusions Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis., Willem Wiersinga and colleagues find up-regulation of multiple Toll-like receptors (TLRs) in peripheral blood cells of patients with melioidosis. However, only TLR2 had an effect on the immune response in a mouse model., Editors' Summary Background. Melioidosis is a severe tropical infection caused by the bacterium Burkholderia pseudomallei. This soil-dwelling pathogen (disease-causing organism) enters the body through cuts, by swallowed contaminated water, or by inhaled contaminated dust. Here, it can cause a severe lung infection or spread into the blood stream and around the body, where it causes widespread inflammation (sepsis) and organ failure. Untreated septic melioidosis is usually fatal. Even with antibiotic therapy, half the people who develop it in Thailand (a hot spot for melioidosis) die. B. pseudomallei is a “gram-negative” bacterium. That is, it is surrounded by a membrane that stops it taking up a stain used to detect bacteria. This membrane contains a molecule called lipopolysaccharide (LPS). Proteins on immune system cells called Toll-like receptors (TLRs), of which there are many, recognize LPS and other surface molecules common to different pathogens and tell the cells to make cytokines. These cytokines stimulate the immune system to kill the pathogen but also cause inflammation, the underlying problem in septic melioidosis and other forms of sepsis. In other words, TLRs are two-edged swords—they provide an essential first-line defense against pathogens, but cause life-threatening inflammation if overstimulated. Why Was This Study Done? It isn't known which TLRs are involved in melioidosis. TLR4 normally detects LPS, but the surface of B. pseudomallei also carries molecules that interact with TLR2. Understanding how B. pseudomallei interacts with TLRs might suggest new, more effective ways to treat septic melioidosis. Better remedies for this disease are badly needed because, as well as the infections it causes in the community, the US Centers for Disease Control and Prevention has identified B. pseudomallei as a potential bioterrorism agent. In this study, the researchers have characterized the expression and function of TLRs in septic melioidosis using human, in vitro (test tube), and animal approaches. What Did the Researchers Do and Find? The researchers isolated monocytes and granulocytes (immune system cells involved in first-line defenses against pathogens) from patients with melioidosis and from healthy people. The patients' cells made more TLR1, TLR2, TLR4, and CD14 (a protein that enhances the activation of immune system cells by LPS) than those of the healthy controls and more of the mRNAs encoding several other TLRs. Next, the researchers tested the ability of heat-killed B. pseudomallei to induce the release of TNFα (a cytokine produced in response to TLR signaling) from macrophages (immune system cells that swallow up pathogens) isolated from wild-type mice and from mice lacking TLR2 or TLR4. Macrophages isolated from wild-type mice made more TNFα than those from TLR2- or TLR4-deficient mice. In addition, a human kidney cell line engineered to express CD14/TLR2 or CD14/TLR4 but not the parent cell line released IL8 (another cytokine) when stimulated with heat-killed B. pseudomallei. Other experiments in these human cell lines showed that LPS purified from B. pseudomallei signals through TLR2 but not through TLR4. Finally, the researchers tested the ability of TLR2- and TLR4-deficient mice to survive after infection with live B. pseudomallei. Compared with TLR4-deficient or wild-type mice, the TLR2-deficient mice had a strong survival advantage, a lower bacterial load, reduced lung inflammation, and less organ damage. What Do These Findings Mean? These findings show that people with melioidosis have increased expression of several TLRs, any one of which might cause the sepsis associated with B. pseudomallei infection. The in vitro findings indicate that TLR2 and TLR4 both contribute to the responsiveness of immune cells to B. pseudomallei in test tubes, but that only TLR2 detects the LPS of this bacterium. This unexpected result—TLR4 normally responds to LPS—might indicate that there is something unique about the LPS of B. pseudomallei. Finally, the survival of TLR2-deficient mice after infection with B. pseudomallei suggests that TLR2-mediated dysregulation of the immune system in response to invasive B. pseudomallei might cause septic melioidosis. Although these results need confirming in people, they suggest that inhibition of TLR2 in combination with antibiotic therapy might improve outcomes for people with melioidosis. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040248. Information is available from the US Centers for Disease Control and Prevention on melioidosis (in English and Spanish) The UK Health Protection Agency provides information for the public and health professionals on melioidosis Wikipedia has pages on melioidosis and on Toll-like receptors (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages) The MedlinePlus encyclopedia contains a page on sepsis (in English and Spanish)

Tuberculous meningitis (TM) is a severe complication of tuberculosis that mainly occurs during childhood. No murine models are available to study this disease. The purpose of the present study was to develop a murine model to investigate the pathogenesis of TM. Mice were intracerebrally injected with Mycobacterium tuberculosis. Bacilli could be cultured from brain homogenates, and, on histopathological examination, all mice were found to have meningeal cellular infiltration. We found elevated levels of chemoattractants for mononuclear phagocytes and neutrophilic granulocytes. This is the first murine model for TM that can be used for research on the host response to TM, in particular the innate immune response.

Mycobacterium tuberculosis and the associated disease tuberculosis are health risks causing many deaths worldwide each year in humans. M. tuberculosis targets dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) to induce immunosuppression, since interaction of DC-SIGN with mycobacterial mannose-capped lipoarabinomannan (ManLAM) induces interleukin (IL)-10 and prevents DC maturation. We investigated the role of a murine homolog of DC-SIGN, SIGN Related 1 (SIGNR1), in a model of M. tuberculosis infection using SIGNR1 deficient (KO) mice. Although SIGNR1 is expressed by macrophages and not by DCs, it also interacts with M. tuberculosis similar to DC-SIGN. Peritoneal macrophages from SIGNR1 KO mice produce less IL-10 upon stimulation with ManLAM than those from wild-type mice, suggesting that the interaction of ManLAM with SIGNR1 can result in immunosuppression similar to its human homolog. Indeed, early in infection, we observed increased T cell activity in SIGNR1 KO mice and increased IFNgamma production by splenocytes in SIGNR1 KO mice. However, we did not detect any differences between WT and KO mice in mycobacterial loads in the lungs or distant organs after M. tuberculosis infection resulting in similar survival rates. Moreover, we found that SIGNR1 is not present on alveolar macrophages of uninfected mice nor is it induced on lung macrophages throughout infection. Therefore, our data suggest that although SIGNR1 has a similar binding specificity as DC-SIGN, its role is limited during murine M. tuberculosis infection.

Background. Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies Toll-like receptor-initiated responses against pathogens. We aimed to characterize TREM-1 expression and function during sepsis caused by Burkholderia pseudomallei (melioidosis). Methods. TREM- 1 expression was determined on leukocytes and plasma from 34 patients with melioidosis and 32 controls and in mice with experimentally induced melioidosis. Responsiveness toward B. pseudomallei of TREM-1 + and TREM-1 - leukocytes was tested in vitro. TREM- 1 function was inhibited in mice by a synthetic peptide mimicking the ectodomain of this receptor. Results. Patients demonstrated increased soluble (s) TREM-1 plasma levels and TREM-1 surface expression on monocytes but not granulocytes. Similarly, mice inoculated with B. pseudomallei displayed a gradual rise in sTREM- 1 level and an increase in blood monocyte but not granulocyte TREM-1 expression. At the primary infection site, however, granulocyte TREM- 1 expression was enhanced, and the rise in sTREM- 1 level occurred earlier. Additionally, purified human TREM-1 - granulocytes showed reduced responsiveness to B. pseudomallei relative to TREM-1 + granulocytes, a difference not detected for TREM-1 - and TREM-1 + monocytes. Treatment with a peptide mimicking a conserved domain of sTREM-1 partially protected mice from B. pseudomallei-induced lethality. Conclusions. During melioidosis, TREM-1 expression is differentially regulated on granulocytes and monocytes; measurement of TREM-1 expression on blood granulocytes may not provide adequate information on granulocyte TREM-1 expression at the infection site. TREM-1 maybe a therapeutic target in melioidosis.

Received 2 January 2007/Returned for modification 12 February 2007/Accepted 16 May 2007 There is an urgent need for an efficacious vaccine against tuberculosis (TB). Cellular immune responses are key to an effective protective response against TB. Recombinant adenovirus (rAd) vectors are especially suited to the induction of strong T-cell immunity and thus represent promising vaccine vehicles for the prevention of TB. We have previously reported on rAd vector serotype 35, the serotype of choice due to low preexisting immunity worldwide, which expresses a unique fusion protein of Mycobacterium tuberculosis antigens Ag85A, Ag85B, and TB10.4 (Ad35-TBS). Here, we demonstrate that Ad35-TBS confers protection against M. tuberculosis when administered to mice through either an intranasal or an intramuscular route. Histological evaluation of lung tissue corroborated the protection and, in addition, demonstrated differences between two mouse strains, with diffuse inflammation in BALB/c mice and distinct granuloma formation in C57BL/6 mice. Epitope mapping analysis in these mouse strains showed that the major T-cell epitopes are conserved in the artificial fusion protein, while three novel CD8 peptides were discovered. Using a defined set of T-cell epitopes, we reveal differences between the two mouse strains in the type of protective immune response, demonstrating that different antigen-specific gamma interferon (IFN-)-producing T cells can provide protection against M. tuberculosis challenge. While in BALB/c (H-2 d ) mice, a dominant CD8 T-cell response was detected, in C57BL/6 (H-2 b ) mice, more balanced CD4/CD8 T-cell responses were observed, with a more pronounced CD4 response in the lungs. These results unify conflicting reports on the relative importance of CD4 versus CD8 T-cell responses in protection and emphasize the key role of IFN-. Tuberculosis (TB), an airborne disease caused by Mycobacterium tuberculosis, is responsible for 2 million deaths each year, with more than 90% of cases occurring in developing countries. It has been estimated that one-third of the world population is infected with M. tuberculosis, and about 5 to 10% of the infected individuals will develop TB during their lifetime. The increasing global impact of TB has been linked to growing poverty, increased emigration, deterioration of public health care, the spread of human immunodeficiency virus, and the development of multidrug-resistant strains of M. tubercu