Bacterial Growth In Lungs Research Articles

Nbeal2 Deficiency Increases Organ Damage but Does Not Affect Host Defense During Gram-Negative Pneumonia-Derived Sepsis.

Objective- Nbeal2-/- mice, a model of human gray platelet syndrome, have reduced neutrophil granularity and impaired host defense against systemic Staphylococcus aureus infection. We here aimed to study the role of Nbeal2 deficiency in both leukocytes and platelets during gram-negative pneumonia and sepsis. Approach and Results- We studied the role of Nbeal2 in platelets and leukocytes during murine pneumonia and sepsis by Klebsiella pneumoniae. Apart from platelet α-granule deficiency and reduced neutrophil granularity, also monocyte granularity was reduced in Nbeal2-/- mice, whereas plasma levels of MPO (myeloperoxidase), elastase, NGAL (neutrophil gelatinase-associated lipocalin), and MMP-9 (matrix metalloproteinase 9), and leukocyte CD11b expression were increased. Nbeal2-/- leukocytes showed unaltered in vitro antibacterial response and phagocytosis capacity against Klebsiella, and unchanged reactive nitrogen species and cytokine production. Also during Klebsiella pneumonia and sepsis, Nbeal2-/- mice had similar bacterial growth in lung and distant body sites, with enhanced leukocyte migration to the bronchoalveolar space. Despite similar infection-induced inflammation, organ damage was increased in Nbeal2-/- mice, which was also seen during endotoxemia. Platelet-specific Nbeal2 deficiency did not influence leukocyte functions, indicating that Nbeal2 directly modifies leukocytes. Transfusion of Nbeal2-/- but not of Nbeal2+/+ platelets into thrombocytopenic mice was associated with bleeding in the lung but similar host defense, pointing at a role for platelet α-granules in maintaining vascular integrity but not host defense during Klebsiella pneumosepsis. Conclusions- These data show that Nbeal2 deficiency-resulting in gray platelet syndrome-affects platelets, neutrophils, and monocytes, with intact host defense but increased organ damage during gram-negative pneumosepsis.

Platelet glycoprotein VI aids in local immunity during pneumonia-derived sepsis caused by gram-negative bacteria

AbstractPlatelet collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type lectin-like receptor 2 (CLEC2) are receptors implicated in platelet activation that both signal via an immunoreceptor tyrosine–based activation motif. Platelets are necessary for host defense and prevention of hemorrhage during sepsis, but the role of platelet GPVI and CLEC2 herein is unknown. To investigate this, we infected mice depleted of platelet GPVI or CLEC2 by antibody treatment or GPVI−/− mice with the common human sepsis pathogen Klebsiella pneumoniae via the airways to induce pneumonia-derived sepsis. The GPVI ligand collagen and the CLEC2 ligand podoplanin were constitutively present in the lung, whereas the GPVI ligands fibrin and histone were induced during pneumonia. During late-stage infection, both mice depleted of GPVI and GPVI−/− mice showed increased bacterial growth in lungs, and GPVI−/− mice also showed increased bacterial growth in distant body sites. Despite higher bacterial loads, GPVI-depleted mice showed reduced platelet numbers, platelet activation, and platelet–leukocyte complex formation in the bronchoalveolar space. Consistently, in human whole blood, GPVI stimulation of platelets increased platelet–leukocyte complex formation and leukocyte activation, which was accompanied by enhanced phagocytosis of Klebsiella. GPVI-depleted mice showed increased lung hemorrhage during infection, but not to the extent observed in platelet-depleted mice, and lung bleeding was not significantly different between GPVI−/− and wild-type mice. CLEC2 depletion did not affect any of the responses during pneumonia. These results suggest that platelet GPVI, but not CLEC2, contributes to local host defense during pneumonia-derived sepsis by enhancing leukocyte function.

Limited role of kininogen in the host response during gram-negative pneumonia-derived sepsis.

High-molecular-weight kininogen (HK), together with factor XI, factor XII and prekallikrein, is part of the contact system that has proinflammatory, prothrombotic, and vasoactive properties. We hypothesized that HK plays a role in the host response during pneumonia-derived sepsis. To this end mice were depleted of kininogen (KNG) to plasma HK levels of 28% of normal by repeated treatment with a specific antisense oligonucleotide (KNG ASO) for 3 wk before infection with the common human sepsis pathogen Klebsiella pneumoniae via the airways. Whereas plasma HK levels increased during infection in mice treated with a scrambled control ASO (Ctrl ASO), HK level in the KNG ASO-treated group remained reduced to 25-30% of that in the corresponding Ctrl ASO group both before and after infection. KNG depletion did not influence bacterial growth in lungs or dissemination to distant body sites. KNG depletion was associated with lower lung CXC chemokine and myeloperoxidase levels but did not impact neutrophil influx, lung pathology, activation of the vascular endothelium, activation of the coagulation system, or the extent of distant organ injury. These results were corroborated by studies in mice with a genetic deficiency of KNG, which were indistinguishable from wild-type mice during Klebsiella-induced sepsis. Both KNG depletion and KNG deficiency were associated with strongly reduced plasma prekallikrein levels, indicating the carrier function of HK for this zymogen. This study suggests that KNG does not significantly contribute to the host defense during gram-negative pneumonia-derived sepsis.

Pseudomonas aeruginosa Exolysin promotes bacterial growth in lungs, alveolar damage and bacterial dissemination

Exolysin (ExlA) is a recently-identified pore-forming toxin secreted by a subset of Pseudomonas aeruginosa strains identified worldwide and devoid of Type III secretion system (T3SS), a major virulence factor. Here, we characterized at the ultrastructural level the lesions caused by an ExlA-secreting strain, CLJ1, in mouse infected lungs. CLJ1 induced necrotic lesions in pneumocytes and endothelial cells, resulting in alveolo-vascular barrier breakdown. Ectopic expression of ExlA in an exlA-negative strain induced similar tissue injuries. In addition, ExlA conferred on bacteria the capacity to proliferate in lungs and to disseminate in secondary organs, similar to bacteria possessing a functional T3SS. CLJ1 did not promote a strong neutrophil infiltration in the alveoli, owing to the weak pro-inflammatory cytokine reaction engendered by the strain. However, CLJ1 was rapidly eliminated from the blood in a bacteremia model, suggesting that it can be promptly phagocytosed by immune cells. Together, our study ascribes to ExlA-secreting bacteria the capacity to proliferate in the lung and to damage pulmonary tissues, thereby promoting metastatic infections, in absence of substantial immune response exacerbation.

Intra-Abdominal Hypertension Causes Bacterial Growth in Lungs: An Animal Study.

To study the effect of intra-abdominal hypertension (IAH) on the frequency of pneumonia with an experimental study, thirteen Sprague-Dawley rats were included. Eight out of thirteen animals were randomly assigned to receive 10 ml of benzalkonium chloride 0.2% (megacolon group) and five animals received 10 ml NaCl 0.9% (controls). Animals were anaesthetized by intramuscular delivery of ketamine. The incidence of positivity for bacteria lung tissue cultures and mesenteric lymph node cultures was assessed at the 21st day after animals' sacrification, or before in case of death. All megacolon group animals presented progressive increase of the abdomen and increased IAP (≥10 mmHg) whereas the frequency of their evacuations was almost eliminated. Controls presented normal evacuations, no sign of abdominal distention, and normal IAP. In megacolon group animals, there was evidence of significant amount of bacteria in lung cultures. In contrast, no bacteria were found in control animals.

Thrombocytopenia impairs host defense in gram-negative pneumonia–derived sepsis in mice

AbstractThrombocytopenia is a common finding in sepsis and associated with a worse outcome. We used a mouse model of pneumonia-derived sepsis caused by the human pathogen Klebsiella pneumoniae to study the role of platelets in host response to sepsis. Platelet counts (PCs) were reduced to less than a median of 5 × 109/L or to 5 to 13 × 109/L by administration of a depleting antibody in mice infected with Klebsiella via the airways. Thrombocytopenia was associated with strongly impaired survival during pneumonia-derived sepsis proportional to the extent of platelet depletion. Thrombocytopenic mice demonstrated PC-dependent enhanced bacterial growth in lungs, blood, and distant organs. Severe thrombocytopenia resulted in hemorrhage at the primary site of infection, but not in distant organs. PCs of 5 to 13 × 109/L were sufficient to largely maintain hemostasis in infected lungs. Thrombocytopenia did not influence lung inflammation or neutrophil recruitment and did not attenuate local or systemic activation of coagulation or the vascular endothelium. PCs <5 × 109/L even resulted in enhanced coagulation and endothelial cell activation, which coincided with increased proinflammatory cytokine levels. In accordance, low PCs in whole blood enhanced Klebsiella-induced cytokine release in vitro. These data suggest that platelets play an important role in host defense to Klebsiella pneumosepsis.

The endothelial protein C receptor impairs the antibacterial response in murine pneumococcal pneumonia and sepsis

Pneumococcal pneumonia is a frequent cause of gram-positive sepsis and has a high mortality. The endothelial protein C receptor (EPCR) has been implicated in both the activation of protein C (PC) and the anti-inflammatory actions of activated (A)PC. The aim of this study was to determine the role of the EPCR in murine pneumococcal pneumonia and sepsis. Wild-type (WT), EPCR knockout (KO) and Tie2-EPCR mice, which overexpress EPCR on the endothelium, were infected intranasally (pneumonia) or intravenously (sepsis) with viable Streptococcus pneumoniae and euthanised at 24 or 48 hours after initiation of the infection for analyses. Pneumonia did not alter constitutive EPCR expression on pulmonary endothelium but was associated with an influx of EPCR positive neutrophils into lung tissue. In pneumococcal pneumonia EPCR KO mice demonstrated diminished bacterial growth in the lungs and dissemination to spleen and liver, reduced neutrophil recruitment to the lungs and a mitigated inflammatory response. Moreover, EPCR KO mice displayed enhanced activation of coagulation in the early phase of disease. Correspondingly, in pneumococcal sepsis EPCR KO mice showed reduced bacterial growth in lung and liver and attenuated cytokine release. Conversely, EPCR-overexpressing mice displayed higher bacterial outgrowth in lung, blood, spleen and liver in pneumococcal sepsis. In conclusion, EPCR impairs antibacterial defense in both pneumococcal pneumonia and sepsis, which is associated with an enhanced pro-inflammatory response.

Host defence duringKlebsiellapneumonia relies on haematopoietic-expressed Toll-like receptors 4 and 2

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.

Intranasal Inoculation of Mice withYersinia pseudotuberculosisCauses a Lethal Lung Infection That Is Dependent onYersiniaOuter Proteins andPhoP

Yersinia pseudotuberculosis infects many mammals and birds including humans, livestock, and wild rodents and can be recovered from the lungs of infected animals. To determine the Y. pseudotuberculosis factors important for growth during lung infection, we developed an intranasal model of infection in mice. Following intranasal inoculation, we monitored both bacterial growth in lungs and dissemination to systemic tissues. Intranasal inoculation with as few as 18 CFU of Y. pseudotuberculosis caused a lethal lung infection in some mice. Over the course of 7 days, wild-type Y. pseudotuberculosis replicated to nearly 1 x 10(8) CFU/g of lung in BALB/c mice, induced histopathology in lungs consistent with pneumonia, but disseminated sporadically to other tissues. In contrast, a Delta yopB deletion strain was attenuated in this model, indicating that translocation of Yersinia outer proteins (Yops) is essential for virulence. Additionally, a Delta yopH null mutant failed to grow to wild-type levels by 4 days postintranasal inoculation, but deletions of any other single effector YOP did not attenuate lung colonization 4 days postinfection. Strains with deletions in yopH and any one of the other known effector yop genes were more attenuated that the Delta yopH strain, indicating a unique role for yopH in lungs. In summary, we have characterized the progression of a lung infection with an enteric Yersinia pathogen and shown that YopB and YopH are important in lung colonization and dissemination. Furthermore, this lung infection model with Y. pseudotuberculosis can be used to test potential therapeutics against Yersinia and other gram-negative infections in lungs.

Pneumococcal behavior and host responses during bronchopneumonia are affected differently by the cytolytic and complement-activating activities of pneumolysin.

Pneumolysin, a multifunctional toxin produced by all clinical isolates of Streptococcus pneumoniae, is strongly implicated in the pathogenesis of pneumococcal bronchopneumonia and septicemia. Using isogenic mutant strains, we examined the effect of deletion of the cytotoxic activity or complement-activating activity of pneumolysin on bacterial growth in lungs and blood, histological changes in infected lung tissue, and the pattern of inflammatory cell recruitment. Both of the activities of pneumolysin contributed to the pathology in the lungs, as well as the timing of the onset of bacteremia. Histological changes in the lungs were delayed after infection with either mutant compared to the changes seen after infection with the wild-type pneumococcus. The complement-activating activity of pneumolysin affected the accumulation of T cells, whereas the toxin's cytolytic activity influenced neutrophil recruitment into lung tissue.

Immunomodulation of pneumococcal pulmonary infection with N(G)-monomethyl-L-arginine.

It has recently become apparent that inflammatory reactions including nitric oxide (NO) release contribute to the outcome of pulmonary infections. To investigate the effect of N(G)-monomethyl-L-arginine (L-NMMA), a NO synthase inhibitor, on the pathogenesis of pneumococcal pneumonia, we inoculated CD(1) Swiss mice with 10(7) CFU of Streptococcus pneumoniae. Treatment with two daily subcutaneous injections of 3 mg of L-NMMA per kg of body weight (over a 5-day period) reproducibly delayed mortality, as the number of surviving mice 72, 84, and 96 h after infection was increased by 16.8% (P < 0.05), 25.0% (P < 0.005), and 11.5% (P < 0. 05), respectively. In fact, the following chronology of events was noted in L-NMMA-treated infected animals, compared to the untreated infected controls. (i) At 12 to 24 h after infection, larger amounts of leukotriene B(4) in bronchoalveolar lavage (BAL) fluid associated with greater neutrophilia in lung tissue and alveolar spaces and more persistent release of tumor necrosis factor alpha, interleukin-1 alpha (IL-1alpha), and IL-6 were observed. (ii) At 24 to 72 h, there was better preservation of lung ultrastructure, including reduction of edema in the interstitium and protection of alveolar spaces, despite identical bacterial growth in lungs, in L-NMMA-treated infected animals than in untreated animals. (iii) At 72 to 96 h, the death rate was delayed, despite the absence of antibiotic therapy. In our experiment, partial blockade of NO release was achieved. These data indicate that NO plays an important role in the induction of tissue injury and death during pneumococcal pneumonia and that L-NMMA is helpful for host protection.