Immunostimulation with macrophage-activating lipopeptide-2 increased survival in murine pneumonia

Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.     

CD14 contributes to pulmonary inflammation and mortality during murine tuberculosis

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.     

Role of Toll-like receptor 4 in gram-positive and gram-negative pneumonia in mice

To determine the role of Toll-like receptor 4 (TLR4) in the immune response to pneumonia, C3H/HeJ mice (which display a mutant nonfunctional TLR4) and C3H/HeN wild-type mice were intranasally infected with either Streptococcus pneumoniae (a common gram-positive respiratory pathogen) or Klebsiella pneumoniae (a common gram-negative respiratory pathogen). In cases of pneumococcal pneumonia, TLR4 mutant mice showed a reduced survival only after infection with low-level bacterial doses, which was associated with a higher bacterial burden in their lungs 48 h postinfection. In Klebsiella pneumonia, TLR4 mutant mice demonstrated a shortened survival after infection with either a low- or a high-level bacterial dose together with an enhanced bacterial outgrowth in their lungs. These data suggest that TLR4 contributes to a protective immune response in both pneumococcal and Klebsiella pneumonia and that its role is more important in respiratory tract infection caused by the latter (gram-negative) pathogen.     

The role of CD44 in the acute and resolution phase of the host response during pneumococcal pneumonia

Streptococcus pneumoniae is the most prevalent pathogen causing community-acquired pneumonia. CD44 is a transmembrane adhesion molecule, expressed by a wide variety of cell types, that has several functions in innate and adaptive immune responses. In this study, we tested the hypothesis that CD44 is involved in the host response during pneumococcal pneumonia. On intranasal infection with a lethal dose of S. pneumoniae CD44-knockout (KO) mice showed a prolonged survival when compared with wild-type mice, which was accompanied by a diminished pulmonary bacterial growth and reduced dissemination to distant body sites. Whereas, proinflammatory cytokine responses and lung pathology were not affected, CD44 deficiency resulted in increased early neutrophil influx into the lung. In separate experiments, we confirmed a detrimental role of CD44 in host defense against pneumococci during sublethal pneumonia, as demonstrated by an improved capacity of CD44 KO mice to clear a low infectious dose. In addition, CD44 appeared important for the resolution of lung inflammation during sublethal pneumonia, as shown by histopathology of lung tissue slides. In conclusion, we show here that CD44 facilitates bacterial outgrowth and dissemination during pneumococcal pneumonia, which in lethal infection results in a prolonged survival of CD44 KO mice. Moreover, during sublethal pneumonia CD44 contributes to the resolution of the inflammatory response.     

Pulmonary Immunostimulation with MALP-2 in Influenza Virus-Infected Mice Increases Survival after Pneumococcal Superinfection

Pulmonary infection with influenza virus is frequently complicated by bacterial superinfection, with Streptococcus pneumoniae being the most prevalent causal pathogen and hence often associated with high morbidity and mortality rates. Local immunosuppression due to pulmonary influenza virus infection has been identified as a major cause of the pathogenesis of secondary bacterial lung infection. Thus, specific local stimulation of the pulmonary innate immune system in subjects with influenza virus infection might improve the host defense against secondary bacterial pathogens. In the present study, we examined the effect of pulmonary immunostimulation with Toll-like receptor 2 (TLR-2)-stimulating macrophage-activating lipopeptide 2 (MALP-2) in influenza A virus (IAV)-infected mice on the course of subsequent pneumococcal superinfection. Female C57BL/6N mice infected with IAV were treated with MALP-2 on day 5 and challenged with S. pneumoniae on day 6. Intratracheal MALP-2 application increased proinflammatory cytokine and chemokine release and enhanced the recruitment of leukocytes, mainly neutrophils, into the alveolar space of IAV-infected mice, without detectable systemic side effects. Local pulmonary instillation of MALP-2 in IAV-infected mice 24 h before transnasal pneumococcal infection considerably reduced the bacterial number in the lung tissue without inducing exaggerated inflammation. The pulmonary viral load was not altered by MALP-2. Clinically, MALP-2 treatment of IAV-infected mice increased survival rates and reduced hypothermia and body weight loss after pneumococcal superinfection compared to those of untreated coinfected mice. In conclusion, local immunostimulation with MALP-2 in influenza virus-infected mice improved pulmonary bacterial elimination and increased survival after subsequent pneumococcal superinfection.     

CD14 plays a limited role during influenza A virus infection in vivo

Influenza A is a single stranded (ss)RNA virus that can cause upper respiratory tract infections that in rare cases may progress to pneumonia. Toll-like receptors (TLRs) and CD14 are receptors which recognize viral proteins and nucleic acid of several viruses. CD14 is required for influenza-induced cytokine production during infection of mouse macrophages. In addition, CD14 was shown to bind ssRNA, suggesting an important role for CD14 during infection with influenza. To investigate the role of CD14 during influenza pneumonia we inoculated WT and CD14 KO mice with a non-lethal dose of a mouse adapted strain of influenza A. CD14 KO mice displayed a reduced viral load in the lungs, 2 and 14 days after infection with influenza. Pulmonary cytokine production in CD14 KO mice was reduced at day 2 and elevated at day 8 compared to WT mice. CD14 deficiency did not influence lymphocyte recruitment or lymphocyte activation in lungs and draining lymph nodes 8 days after infection. These data show that CD14 plays a limited role in host defense against infection with influenza.     

Toll-Like Receptor 4 Agonistic Antibody Promotes Innate Immunity against Severe Pneumonia Induced by Coinfection with Influenza Virus and Streptococcus pneumoniae

Coinfection with bacteria is a major cause of mortality during influenza epidemics. Recently, Toll-like receptor (TLR) agonists were shown to have immunomodulatory functions. In the present study, we investigated the effectiveness and mechanisms of the new TLR4 agonistic monoclonal antibody UT12 against secondary pneumococcal pneumonia induced by coinfection with influenza virus in a mouse model. Mice were intranasally inoculated with Streptococcus pneumoniae 2 days after influenza virus inoculation. UT12 was intraperitoneally administered 2 h before each inoculation. Survival rates were significantly increased and body weight loss was significantly decreased by UT12 administration. Additionally, the production of inflammatory mediators was significantly suppressed by the administration of UT12. In a histopathological study, pneumonia in UT12-treated mice was very mild compared to that in control mice. UT12 increased antimicrobial defense through the acceleration of macrophage recruitment into the lower respiratory tract induced by c-Jun N-terminal kinase (JNK) and nuclear factor kappaB (NF-κB) pathway-dependent monocyte chemoattractant protein 1 (MCP-1) production. Collectively, these findings indicate that UT12 promoted pulmonary innate immunity and may reduce the severity of severe pneumonia induced by coinfection with influenza virus and S. pneumoniae. This immunomodulatory effect of UT12 improves the prognosis of secondary pneumococcal pneumonia and makes UT12 an attractive candidate for treating severe infectious diseases.     

Respiratory tract infection with Mycoplasma pneumoniae in interleukin-12 knockout mice results in improved bacterial clearance and reduced pulmonary inflammation

Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentration in respiratory secretions. We evaluated the microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in IL-12 (p35) knockout (KO) mice and wild-type (WT) mice to determine the role of IL-12 in M. pneumoniae respiratory disease. Eight-week-old wild-type BALB/c mice and 8-week-old IL-12 (p35) KO BALB/c mice were inoculated once intranasally with 10(7) CFU of M. pneumoniae. Mice were evaluated at days 2, 4, and 7 after inoculation. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic scores (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) and plethysmography, before and after methacholine, to assess airway obstruction (AO) and airway hyperreactivity (AHR). IL-12 (p35) KO mice infected with M. pneumoniae were found to have significantly lower BAL M. pneumoniae concentrations compared with M. pneumoniae-infected WT mice. Lung HPS and the parenchymal pneumonia subscores (neutrophilic alveolar infiltrate), as well as AO, were significantly lower in infected KO mice. No difference was found for AHR. Infected KO mice had significantly lower BAL concentrations of IFN-gamma than WT mice; a trend toward lower BAL concentrations was observed for IL-10 (P = 0.065) and TNF-alpha (P = 0.078). No differences were found for IL-1beta, IL-2, IL-4, IL-5, or IL-6. The lack of IL-12 in experimental M. pneumoniae pneumonia was associated with less severe pulmonary disease and more rapid microbiologic and histologic resolution.     

Leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia

The adipocyte-derived hormone leptin is an important regulator of appetite and energy expenditure and is now appreciated for its ability to control innate and adaptive immune responses. We have reported previously that the leptin-deficient ob/ob mouse exhibited increased susceptibility to the Gram-negative bacterium Klebsiella pneumoniae. In this report we assessed the impact of chronic leptin deficiency, using ob/ob mice, on pneumococcal pneumonia and examined whether restoring circulating leptin to physiological levels in vivo could improve host defences against this pathogen. We observed that ob/ob mice, compared with wild-type (WT) animals, exhibited enhanced lethality and reduced pulmonary bacterial clearance following Streptococcus pneumoniae challenge. These impairments in host defence in ob/ob mice were associated with elevated levels of lung tumour necrosis factor (TNF)-alpha, macrophage inflammatory peptide (MIP)-2 [correction added after online publication 28 September 2007: definition of MIP corrected], prostaglandin E(2) (PGE(2)), lung neutrophil polymorphonuclear leukocyte (PMN) counts, defective alveolar macrophage (AM) phagocytosis and PMN killing of S. pneumoniae in vitro. Exogenous leptin administration to ob/ob mice in vivo improved survival and greatly improved pulmonary bacterial clearance, reduced bacteraemia, reconstituted AM phagocytosis and PMN H(2)O(2) production and killing of S. pneumoniae in vitro. Our results demonstrate, for the first time, that leptin improves pulmonary bacterial clearance and survival in ob/ob mice during pneumococcal pneumonia. Further investigations are warranted to determine whether there is a potential therapeutic role for this adipokine in immunocompromised patients.     

Modulation of the inflammatory response to Streptococcus pneumoniae in a model of acute lung tissue infection

Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia and is a main cause of infectious deaths. However, little is known about host-pathogen interaction in human lung tissue. We tested the hypothesis that human alveolar macrophages (AMs) and alveolar epithelial cells (AECs) are important for initiating the host response against S. pneumoniae, and we evaluated the role of Toll-like receptor (TLR) 2, TLR4, and p38 mitogen-activated protein kinase (MAPK) signaling in the inflammatory response after pneumococcal infection. We established a novel model of acute S. pneumoniae infection using vital human lung specimens. In situ hybridization analysis showed that S. pneumoniae DNA was detected in 80 to 90% of AMs and 15 to 30% of AECs after in vitro infection accompanied by increased expression of inflammatory cytokines. Enhanced phosphorylation of p38 MAPK and increased TLR2 and 4 mRNA expression were observed in infected lung tissue. Thirty to fifty percent of AMs and 10 to 20% of AECs showed evidence of apoptosis 24 hours after pneumococcal infection. After macrophage deactivation with Clodronate/liposomes, infected lung tissue exhibited a significantly decreased release of inflammatory mediators. Inhibition of p38 MAPK signaling markedly reduced inflammatory cytokine release from human lungs, whereas TLR2 blockade revealed only minor effects. AMs are central resident immune cells during S. pneumoniae infection and are the main source of early proinflammatory cytokine release. p38 MAPK holds a major role in pathogen-induced pulmonary cytokine release and is a potential molecular target to modulate overwhelming lung inflammation.     

Impact of endogenous protein C on pulmonary coagulation and injury during lethal H1N1 influenza in mice

Influenza accounts for 5-10% of community-acquired pneumonia cases, and is a major cause of mortality. Sterile and bacterial lung injury are associated with procoagulant and inflammatory derangements in the lungs and down-regulation of the protein C (PC) pathway has been correlated with disease severity and mortality in severe bacterial pneumonia and sepsis. In addition, during lethal influenza pneumonia, pulmonary and systemic coagulation are activated, which can be attenuated by the administration of recombinant activated (A) PC. We here determined the role of endogenous PC in lethal H1N1 influenza A infection. Male C57BL/6 mice pretreated with an inhibitory monoclonal antibody directed against murine PC or a control antibody were intranasally infected with a lethal dose of a mouse-adapted H1N1 influenza A strain. Mice were killed at 48 or 96 hours after infection, after which lungs and bronchoalveolar lavage fluid were harvested, or observed for up to 9 days. Anti-PC antibody treatment aggravated pulmonary activation of coagulation as compared with control antibody treatment, as reflected by increased lung concentrations of thrombin-antithrombin complexes and fibrin degradation products, as well as intravascular thrombus formation. Anti-PC antibody treatment aggravated lung histopathology, but lowered bronchoalveolar neutrophil influx and total protein levels, and delayed mortality. In conclusion, endogenous PC has strong effects on the host response to lethal influenza A infection, inhibiting pulmonary coagulopathy and inflammation on the one hand, but facilitating neutrophil influx and protein leak and accelerating mortality on the other hand.     

CD27 contributes to the early systemic immune response to Mycobacterium tuberculosis infection but does not affect outcome

The development of a strong Th1-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 IFNgamma production by CD4+ and CD8+ lymphocytes in the lungs early after infection, the number of splenic CD8+ T cells producing the key Th1 cytokine IFNgamma 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 IFNgamma-mediated response and long-term protection against M. tuberculosis.     

Monocyte chemoattractant protein 1 contributes to an adequate immune response in influenza pneumonia

Monocyte chemoattractant protein 1 (MCP-1) and its receptor CCR2 have been shown to play an import role in leukocyte recruitment to sites of infection and inflammation. To investigate the role of MCP-1 during infection with influenza we inoculated wild-type (WT) and MCP-1 knockout (KO) mice with a non-lethal dose of a mouse adapted strain of influenza A. Influenza infection of WT mice resulted in a profound increase in pulmonary MCP-1 levels. MCP-1 KO mice had enhanced weight loss and did not fully regain their body weight during the 14-day observation period. In addition, MCP-1 KO mice demonstrated elevated viral loads 8 days after infection, which was accompanied by reduced leukocyte recruitment into the infected lungs, primarily caused by a diminished influx of macrophages and granulocytes. Moreover, pulmonary levels of IgA were reduced in MCP-1 KO mice. The pulmonary concentrations of tumor necrosis factor-alpha, interleukin-6, macrophage inflammatory protein 2 and interferon-gamma were higher in MCP-1 KO mice. This study shows that MCP-1 contributes to an adequate protective immune response against influenza infection in mice.     

Critical role of Valpha14+ natural killer T cells in the innate phase of host protection against Streptococcus pneumoniae infection

The present study was designed to elucidate the role of Valpha14(+) NKT cells in the host defense against pulmonary infection with Streptococcus pneumoniae using Jalpha281 gene-disrupted mice (Jalpha281KO mice) that lacked this lymphocyte subset. In these mice, pneumococcal infection was severely exacerbated, as shown by the shorter survival time and marked increase of live bacteria in the lung compared to wild-type (WT) mice. The proportion of Valpha14(+) NKT cells, detected by an alpha-galactosylceramide (alpha-GalCer)-loaded CD1d tetramer, increased in thelung after S. pneumoniae infection. This increase was significantly reduced in mice with a genetic disruption of monocyte chemotactic protein (MCP)-1, which was produced in the early phaseof infection in WT mice. In the lungs of Jalpha281KO mice, the number of neutrophils was significantly lower at 12 h than that in WT mice. In support of this finding, macrophage inflammatory protein (MIP)-2 and TNF-alpha synthesis in infected lungs was significantly reduced at 3 h and at both 3 and 6 h, respectively, in Jalpha281KO mice, compared to WT mice. In addition, treatment of mice with alpha-GalCer significantly improved the outcome of this infection. Our results demonstrated MCP-1-dependent recruitment of Valpha14(+) NKT cells and their critical role in early host protection against S. pneumoniae by promoting the trafficking of neutrophils to the site of infection.     

Tracheal function during influenza infections.

Studies with animal models have demonstrated that viral respiratory tract infections suppress bacterial clearance processes in the lung and that this alteration in host defenses appears to explain the excessive mortality from bacterial pneumonia during influenza epidemics. However, since the pathogenesis of postinfluenza pneumonia and other pneumonias probably involves the aspiration of normal nasopharyngeal flora, injury to major airways associated with influenza infections could also contribute to the development of bacterial pneumonia by increasing bacterial deposition in the peripheral lung. We investigated this possibility by evaluating tracheal clearance processes and spontaneous changes in the tracheal flora in a murine model for acute influenza. During fine-particle aerosol exposures to Staphylococcus aureus, influenza-infected mice retained the same number of bacteria on their proximal tracheal surfaces as did control mice, and the relative adherence of the staphylococci to the trachea was similar in both groups of mice. However, the clearance of viable staphylococci from the trachea was significantly delayed in influenza-infected mice. Control and influenza-infected mice were also evaluated for changes in their normal tracheal flora. Mice with established influenza infections had more frequent spontaneous colonization with gram-negative bacteria, more bacterial isolates per animal, and higher bacterial concentrations in tracheal homogenates than control mice. These changes in tracheal flora were most pronounced on day 7 after virus inoculation and persisted after virus titers were undetectable, but eventually resolved by day 14 after virus infection. Tetracycline therapy started 2 days after virus inoculation prevented the increased colonization. This impaired clearance function and increased spontaneous colonization were associated with morphological evidence of mucosal regeneration. We conclude that spontaneous changes in tracheal flora occur during influenza infections, that these changes reflect, in part, impaired clearance functions, and that such changes could contribute to the development of pneumonia regardless of the clearance capacity of the lung parenchyma.     

LPS-binding protein-deficient mice have an impaired defense against Gram-negative but not Gram-positive pneumonia

LPS-binding protein (LBP) can facilitate the transfer of cell wall components of both Gram-negative bacteria (LPS) and Gram-positive bacteria (lipoteichoic acid) to inflammatory cells. Although LBP is predominantly produced in the liver, recent studies have indicated that this protein is also synthesized locally in the lung by epithelial cells. To determine the role of LBP in the immune response to pneumonia, LBP gene-deficient (-/-) and normal wild-type (WT) mice were intra-nasally infected with either Streptococcus pneumoniae or Klebsiella pneumoniae, common Gram-positive and Gram-negative pathogens, respectively. Pneumococcal pneumonia was associated with a 7-fold rise in LBP concentrations in bronchoalveolar lavage fluid of WT mice; LBP-/- mice infected with S. pneumoniae showed a similar survival and a similar bacterial burden in their lungs 48 h post-infection. In Klebsiella pneumonia, however, LBP-/- mice demonstrated a diminished survival together with an enhanced bacterial outgrowth in their lungs. These data suggest that LBP is important for a protective immune response in Klebsiella pneumonia, but does not contribute to an effective host response in pneumococcal pneumonia.     

Tumor necrosis factor as a mediator of inflammation in influenza A viral pneumonia

The role of tumor necrosis factor α (TNFα) in the pathogenesis of influenza A viral pneumonia was examined. CD-1 male mice were challenged intranasally with influenza A virus A/PR/8/34 (H1N1) and administered rabbit anti-mouse TNFα-specific-neutralizing antibodies intraperitoneally. The effect of treatment on virus titer, TNFα levels, morbidity, mortality, and on pathologic lung lesions were compared with sham-treated controls. The severity of gross and histologic lung lesions positively correlated with the peak bronchoalveolar TNFα levels and was ameliorated with anti-TNFα treatment. Survivorship was prolonged in mice given a lethal dose of virus by treatment with TNF-α neutralizing antibodies. Reduction of TNFα levels by treatment with TNFα-antibodies did not affect virus titers in the lung. These results suggest that TNFα is a mediator of pulmonary inflammation during influenza A viral pneumonia, but may not play a significant anti-viral role in influenza pneumonia.     

Role of invariant NKT cells in lipopolysaccharide-induced lethal shock during encephalomyocarditis virus infection

Viral infections can give rise to secondary bacterial infections. In the present study, we examined the role of invariant natural killer T (iNKT) cells in lipopolysaccharide (LPS)-induced lethal shock during encephalomyocarditis virus (EMCV) infection. Wild-type (WT) mice and Jα18 gene knockout (Jα18 KO) mice were inoculated with EMCV, 5 days prior to challenging with LPS. The survival rate of Jα18 KO mice subjected to EMCV and LPS was significantly higher than that of WT mice. TNF-α and nitric oxide (NO) production were increased in WT mice, than that in Jα18 KO mice, after the administration of EMCV and LPS. EMCV infection increased the number of iNKT cells and IFN-γ production by iNKT cells in WT mice. Moreover, EMCV infection enhanced the expression of Toll-like receptor 4 (TLR4) in the lung and spleen. IFN-γ also increased the expression of TLR4 in splenocytes. These findings indicated that EMCV infection activated iNKT cells, and IFN-γ secreted from the iNKT cells up-regulated the expression of TLR4 in various tissues. As a result, EMCV-infected mice were susceptible to LPS and easily developed the lethal shock. In conclusion, iNKT cells were involved in the development of LPS-induced lethal shock during EMCV infection.     

Myeloid differentiation protein-2-dependent and -independent neutrophil accumulation during Escherichia coli pneumonia

Bacterial pneumonia remains a serious disease. Pattern recognition receptors play an integral role in neutrophil accumulation during pneumonia. Although myeloid differentiation protein (MD)-2 has been recognized as a key molecule for LPS signaling, the role of MD-2 in neutrophil accumulation in the lung during bacterial infection has not been explored. Here, we investigate the role of MD-2 in Escherichia coli LPS-induced lung inflammation and E. coli-induced pneumonia. LPS-induced CD14-independent neutrophil accumulation was abolished in CD14/MD-2(-/-) mice. MD-2(-/-) mice challenged with LPS displayed attenuated neutrophil influx, NF-kappaB activation, cytokine/chemokine expression, and lung histopathology. MD-2(-/-) mice transplanted with MD-2(+/+) bone marrow demonstrated decreased neutrophil influx and cytokine/chemokine expression in the lungs when challenged by LPS. MD-2(-/-) mice infected with E. coli demonstrated reduced neutrophil influx and cytokine/chemokine expression in the lungs, whereas heat-killed E. coli did not induce either neutrophil accumulation or cytokine/chemokine expression in MD-2(-/-) mice infected with E. coli. Furthermore, MD-2(-/-) mice displayed increased bacterial burden in the lungs and enhanced bacterial dissemination. Toll-like receptor (TLR)-5(-/-) mice infected with E. coli exhibited attenuated neutrophil accumulation, whereas MD-2/TLR5(-/-) mice inoculated with E. coli showed further attenuated neutrophil influx and impaired bacterial clearance. Taken together, these new findings demonstrate: (1) the important role of MD-2 in the CD14-independent LPS-mediated cascade of neutrophil influx; (2) the relative importance of bone marrow- and non-bone marrow cell-derived MD-2 in LPS-induced inflammation; and (3) the essential role of MD-2-dependent and MD-2-independent (TLR5) signaling in E. coli-induced neutrophil accumulation and pulmonary host defense.