Pneumonia-derived Sepsis Research Articles

Clinical application value of simultaneous plasma and bronchoalveolar lavage fluid metagenomic next generation sequencing in patients with pneumonia-derived sepsis

BackgroundDespite the increasing use of metagenomic next-generation sequencing (mNGS) in sepsis, identifying clinically relevant pathogens remains challenging. This study was aimed to evaluate the clinical utility of simultaneous plasma and bronchoalveolar lavage fluid (BALF) detection using mNGS.MethodsThis retrospective study enrolled 95 patients with pneumonia-derived sepsis (PDS) admitted to the intensive care unit (ICU) between October 2021 and January 2023. Patients were divided into two groups: mNGS group (n = 60) and the non-mNGS group (n = 35), based on whether simultaneous plasma and BALF mNGS were conducted. All patients underwent conventional microbiological tests (CMT), including bacterial/fungal culture of peripheral blood and BALF, as well as sputum culture, detection of 1, 3-beta-D- glucan in BALF and RT-PCR testing. The clinical data of the enrolled patients were collected, and the detection performance and prognosis of plasma mNGS, BALF mNGS and CMT were compared.ResultsThe mNGS group exhibited a lower mortality rate than the non-mNGS group (35.0% vs. 57.1%, P = 0.034). Simultaneous detection in dual-sample resulted in a higher proportion of microorganisms identified as definite causes of sepsis alert compared to detection in either plasma or BALF alone (55.6% vs. 20.8% vs. 18.8%, P<0.001). Acinetobacter baumannii, Stenotrophomonas maltophilia, Candida albicans, and human mastadenovirus B were the primary strains responsible for infections in PDS patients. Patients with lower white blood cells and neutrophil indices had a greater consistency in dual-sample mNGS. Patients in the mNGS group had more antibiotic adjustments compared to the non-mNGS group (85.71% vs. 33.33%, P<0.001). The percentage of neutrophils was a risk factor for mortality in PDS patients (P = 0.002).ConclusionDual sample mNGS has the advantage of detecting and determining the pathogenicity of more pathogens and has the potential to improve the prognosis of patients with PDS.

Gut microbes improve prognosis of Klebsiella pneumoniae pulmonary infection through the lung-gut axis.

The gut microbiota plays a vital role in the development of sepsis and in protecting against pneumonia. Previous studies have demonstrated the existence of the gut-lung axis and the interaction between the gut and the lung, which is related to the prognosis of critically ill patients; however, most of these studies focused on chronic lung diseases and influenza virus infections. The purpose of this study was to investigate the effect of faecal microbiota transplantation (FMT) on Klebsiella pneumoniae-related pulmonary infection via the gut-lung axis and to compare the effects of FMT with those of traditional antibiotics to identify new therapeutic strategies. We divided the mice into six groups: the blank control (PBS), pneumonia-derived sepsis (KP), pneumonia-derived sepsis + antibiotic (KP + PIP), pneumonia-derived sepsis + faecal microbiota transplantation(KP + FMT), antibiotic treatment control (KP+PIP+PBS), and pneumonia-derived sepsis+ antibiotic + faecal microbiota transplantation (KP + PIP + FMT) groups to compare the survival of mice, lung injury, inflammation response, airway barrier function and the intestinal flora, metabolites and drug resistance genes in each group. Alterations in specific intestinal flora can occur in the gut of patients with pneumonia-derived sepsis caused by Klebsiella pneumoniae. Compared with those in the faecal microbiota transplantation group, the antibiotic treatment group had lower levels of proinflammatory factors and higher levels of anti-inflammatory factors but less amelioration of lung pathology and improvement of airway epithelial barrier function. Additionally, the increase in opportunistic pathogens and drug resistance-related genes in the gut of mice was accompanied by decreased production of favourable fatty acids such as acetic acid, propionic acid, butyric acid, decanoic acid, and secondary bile acids such as chenodeoxycholic acid 3-sulfate, isodeoxycholic acid, taurodeoxycholic acid, and 3-dehydrocholic acid; the levels of these metabolites were restored by faecal microbiota transplantation. Faecal microbiota transplantation after antibiotic treatment can gradually ameliorate gut microbiota disorder caused by antibiotic treatment and reduce the number of drug resistance genes induced by antibiotics. In contrast to direct antibiotic treatment, faecal microbiota transplantation improves the prognosis of mice with pneumonia-derived sepsis caused by Klebsiella pneumoniae by improving the structure of the intestinal flora and increasing the level of beneficial metabolites, fatty acids and secondary bile acids, thereby reducing systemic inflammation, repairing the barrier function of alveolar epithelial cells, and alleviating pathological damage to the lungs. The combination of antibiotics with faecal microbiota transplantation significantly alleviates intestinal microbiota disorder, reduces the selection for drug resistance genes caused by antibiotics, and mitigates lung lesions; these effects are superior to those following antibiotic monotherapy.

Time-resolved RNA-seq analysis to unravel the in vivo competence induction by Streptococcus pneumoniae during pneumonia-derived sepsis.

Competence development in Streptococcus pneumoniae (pneumococcus) is tightly intertwined with virulence. In addition to genes encoding genetic transformation machinery, the competence regulon also regulates the expression of allolytic factors, bacteriocins, and cytotoxins. Pneumococcal competence system has been extensively interrogated in vitro where the short transient competent state upregulates the expression of three distinct phases of "early," "late," and "delayed" genes. Recently, we have demonstrated that the pneumococcal competent state develops naturally in mouse models of pneumonia-derived sepsis. To unravel the underlying adaptive mechanisms driving the development of the competent state, we conducted a time-resolved transcriptomic analysis guided by the spatiotemporal live in vivo imaging system of competence induction during pneumonia-derived sepsis. Mouse lungs infected by the serotype 2 strain D39 expressing a competent state-specific reporter gene (D39-ssbB-luc) were subjected to RNA sequencing guided by monitoring the competence development at 0, 12, 24, and, at the moribund state, >40 hours post-infection (hpi). Transcriptomic analysis revealed that the competence-specific gene expression patterns in vivo were distinct from those under in vitro conditions. There was significant upregulation of early, late, and some delayed phase competence-specific genes as early as 12 hpi, suggesting that the pneumococcal competence regulon is important for adaptation to the lung environment. Additionally, members of the histidine triad (pht) gene family were sharply upregulated at 12 hpi followed by a steep decline throughout the rest of the infection cycle, suggesting that Pht proteins participate in the early adaptation to the lung environment. Further analysis revealed that Pht proteins execute a metal ion-dependent regulatory role in competence induction.IMPORTANCEThe induction of pneumococcal competence for genetic transformation has been extensively studied in vitro but poorly understood during lung infection. We utilized a combination of live imaging and RNA sequencing to monitor the development of a competent state during acute pneumonia. Upregulation of competence-specific genes was observed as early as 12 hour post-infection, suggesting that the pneumococcal competence regulon plays an important role in adapting pneumococcus to the stressful lung environment. Among others, we report novel finding that the pneumococcal histidine triad (pht) family of genes participates in the adaptation to the lung environment and regulates pneumococcal competence induction.

Effects of Chengqi Decoction on Complications and Prognosis of Patients with Pneumonia-Derived Sepsis: Retrospective Cohort Study.

Purpose A specific and efficacious method for treatment of pneumonia-derived sepsis is lacking. Chengqi decoction has been used for treatment of pneumonia-derived sepsis, but a clinical trial on patients with pneumonia-derived sepsis is lacking, a gap in the literature that we sought to fill. Patients and Methods. 282 patients with pneumonia-derived sepsis admitted to the intensive care unit of our hospital were selected. They were divided into the treatment group (141 cases) and control group (141 cases). Both groups underwent conventional treatment, but Chengqi decoction (in the form of enema) was given to the treatment group. Mortality, morbidity (abdominal distension and gastrointestinal bleeding), duration of antibiotic use, and use of vasoactive agents were documented 28 days after the drug was used. Results The treatment group reduced mortality and morbidity (abdominal distension) (P < 0.05). After adjustment for significant covariates, 28-day survival was similar for the whole group (hazard ratio (HR): 0.48; 95% confidence interval (CI): 0.23–0.97; P=0.037), for the subgroup (n = 120) with Acute Physiology and Chronic Health Evaluation II score ≥25 (HR: 0.180; 95% CI: 0.032–0.332; P=0.039) and for the subgroup (n = 66) with N-terminal B-type natriuretic peptide <1800 (0.059, 0.004–0.979, and 0.019). There was no difference between the two groups for the duration of antibiotic use, major bleeding, or use of vasoactive drugs. Conclusions Chengqi decoction improved 28-day survival and reduced the prevalence of abdominal distension in patients with pneumonia-derived sepsis.

Pulmonary and intestinal microbiota dynamics during Gram-negative pneumonia-derived sepsis

BackgroundThe gut microbiome plays a protective role in the host defense against pneumonia. The composition of the lung microbiota has been shown to be predictive of clinical outcome in critically ill patients. However, the dynamics of the lung and gut microbiota composition over time during severe pneumonia remains ill defined. We used a mouse model of pneumonia-derived sepsis caused by Klebsiella pneumoniae in order to follow the pathogen burden as well as the composition of the lung, tongue and fecal microbiota from local infection towards systemic spread.ResultsAlready at 6 h post-inoculation with K. pneumoniae, marked changes in the lung microbiota were seen. The alpha diversity of the lung microbiota did not change throughout the infection, whereas the beta diversity did. A shift between the prominent lung microbiota members of Streptococcus and Klebsiella was seen from 12 h onwards and was most pronounced at 18 h post-inoculation (PI) which was also reflected in the release of pro-inflammatory cytokines indicating severe pulmonary inflammation. Around 18 h PI, K. pneumoniae bacteremia was observed together with a systemic inflammatory response. The composition of the tongue microbiota was not affected during infection, even at 18–30 h PI when K. pneumoniae had become the dominant bacterium in the lung. Moreover, we observed differences in the gut microbiota during pulmonary infection. The gut microbiota contributed to the lung microbiota at 12 h PI, however, this decreased at a later stage of the infection.ConclusionsAt 18 h PI, K. pneumoniae was the dominant member in the lung microbiota. The lung microbiota profiles were significantly explained by the lung K. pneumoniae bacterial counts and Klebsiella and Streptococcus were correlating with the measured cytokine levels in the lung and/or blood. The oral microbiota in mice, however, was not influenced by the severity of murine pneumonia, whereas the gut microbiota was affected. This study is of significance for future studies investigating the role of the lung microbiota during pneumonia and sepsis.

NLRP6 modulates neutrophil homeostasis in bacterial pneumonia-derived sepsis.

Bacterial pneumonia is a significant cause of morbidity, mortality, and health care expenditures. Optimum neutrophil recruitment and their function are critical defense mechanisms against respiratory pathogens. The nucleotide-binding oligomerization domain-like receptor (NLRP) 6 controls gut microbiota and immune response to systemic and enteric infections. However, the importance of NLRP6 in neutrophil homeostasis following lung infection remains elusive. To investigate the role of NLRs in neutrophil homeostasis, we used Nlrp6 gene-deficient (Nlrp6−/−) mice in a model of Klebsiella pneumoniae-induced pneumonia-derived sepsis. We demonstrated that NLRP6 is critical for host survival, bacterial clearance, neutrophil influx, and CXC-chemokine production. Kp-infected Nlrp6−/− mice have reduced numbers of hematopoietic stem cells and granulocyte-monocyte progenitors but increased retention of matured neutrophils in bone marrow. Neutrophil extracellular trap (NET) formation and NET-mediated bacterial killing were also impaired in Nlrp6−/− neutrophils in vitro. Furthermore, recombinant CXCL1 rescued the impaired host defense, granulopoietic response, and NETosis in Kp-infected Nlrp6−/− mice. Using A/J background mice and co-housing experiments, our findings revealed that the susceptible phenotype of Nlrp6−/− mice is not strain-specific and gut microbiota-dependent. Taken together, these data unveil NLRP6 as a central regulator of neutrophil recruitment, generation and function during bacterial pneumonia followed by sepsis.

Alterations in the Gut Microbiome and Cecal Metabolome During Klebsiella pneumoniae-Induced Pneumosepsis.

Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis in human and is associated with high morbidity and mortality. The microbiota promotes and maintains host immune homeostasis during bacterial infections. However, the mechanisms by which the gut microbiota affects immune responses in the lung still remain poorly understood. Here, we performed cecal metabolomics sequencing and fecal 16s rRNA sequencing in K. pneumoniae-infected mice and uninfected controls and showed that K. pneumoniae infection led to profound alterations in the gut microbiome and thus the cecal metabolome. We observed that the levels of Lactobacillus reuteri and Bifidobacterium pseudolongum were significantly decreased in K. pneumoniae-infected mice. Spearman correlation analysis showed that alterations in the richness and composition of the gut microbiota were associated with profound changes in host metabolite concentrations. Further, short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, were detected in cecal contents and serum by gas chromatography-mass spectrometry (GC-MS). We observed that the concentrations of these three SCFAs were all lower in the infected groups than in the untreated controls. Lastly, oral supplementation with these three SCFAs reduced susceptibility to K. pneumoniae infections, as indicated by lower bacterial burdens in the lung and higher survival rates. Our data highlight the protective roles of gut microbiota and certain metabolites in K. pneumoniae-pneumonia and suggests that it is possible to intervene in this bacterial pneumonia by targeting the gut microbiota.

Streptococcus pneumoniae Elaborates Persistent and Prolonged Competent State during Pneumonia-Derived Sepsis.

The competence regulon of pneumococcus regulates both genetic transformation and virulence. However, competence induction during host infection has not been examined. By using the serotype 2 strain D39, we transcriptionally fused the firefly luciferase (luc) to competence-specific genes and spatiotemporally monitored the competence development in a mouse model of pneumonia-derived sepsis. In contrast to the universally reported short transient burst of competent state in vitro, the naturally developed competent state was prolonged and persistent during pneumonia-derived sepsis. The competent state began at approximately 20 h postinfection (hpi) and facilitated systemic invasion and sepsis development and progressed in different manners. In some mice, acute pneumonia quickly led to sepsis and death, accompanied by increasing intensity of the competence signal. In the remaining mice, pneumonia lasted longer, with the competence signal decreasing at first but increasing as the infection became septic. The concentration of pneumococcal inoculum (1 × 106 to 1 × 108 CFU/mouse) and postinfection lung bacterial burden did not appreciably impact the kinetics of competence induction. Exogenously provided competence stimulating peptide 1 (CSP1) failed to modulate the onset kinetics of competence development in vivo The competence shutoff regulator DprA was highly expressed during pneumonia-derived sepsis but failed to turn off the competent state in mice. Competent D39 bacteria propagated the competence signal through cell-to-cell contact rather than the classically described quorum-sensing mechanism. Finally, clinical pneumococcal strains of different serotypes were also able to develop natural competence during pneumonia-derived sepsis.

CXCL1 regulates neutrophil homeostasis in pneumonia-derived sepsis caused by Streptococcus pneumoniae serotype 3

AbstractNeutrophil migration to the site of bacterial infection is a critical step in host defense. Exclusively produced in the bone marrow, neutrophil release into the blood is tightly controlled. Although the chemokine CXCL1 induces neutrophil influx during bacterial infections, its role in regulating neutrophil recruitment, granulopoiesis, and neutrophil mobilization in response to lung infection-induced sepsis is unclear. Here, we used a murine model of intrapulmonary Streptococcus pneumoniae infection to investigate the role of CXCL1 in host defense, granulopoiesis, and neutrophil mobilization. Our results demonstrate that CXCL1 augments neutrophil influx to control bacterial growth in the lungs, as well as bacterial dissemination, resulting in improved host survival. This was shown in Cxcl1−/− mice, which exhibited defective amplification of early neutrophil precursors in granulocytic compartments, and CD62L- and CD49d-dependent neutrophil release from the marrow. Administration of recombinant CXCL2 and CXCL5 after infection rescues the impairments in neutrophil-dependent host defense in Cxcl1−/− mice. Taken together, these findings identify CXCL1 as a central player in host defense, granulopoiesis, and mobilization of neutrophils during Gram-positive bacterial pneumonia-induced sepsis.

Bradykinin receptor deficiency or antagonism do not impact the host response during gram-negative pneumonia-derived sepsis

BackgroundKinins are short peptides with a wide range of proinflammatory properties that are generated from kininogens in the so-called kallikrein-kinin system. Kinins exert their biological activities through stimulation of two distinct receptor subtypes, the kinin or bradykinin B1 and B2 receptors (B1R, B2R). Acute challenge models have implicated B1R and B2R in the pathogenesis of sepsis. However, their role in the host response during sepsis originating from the lung is not known.ResultsTo determine the role of B1R and B2R in pneumonia-derived sepsis, B1R/B2R-deficient mice and wild-type mice treated with the B1R antagonist R-715 or the B2R antagonist HOE-140 were studied after infection with the common gram-negative pathogen Klebsiella pneumoniae via the airways. Neither B1R/B2R deficiency nor B1R or B2R inhibition influenced bacterial growth at the primary site of infection or dissemination to distant body sites. In addition, B1R/B2R deficiency or inhibition did not impact local or systemic inflammatory responses during Klebsiella induced pneumosepsis.ConclusionsThese data argue against an important role for kinins in the host response to pneumonia-derived sepsis caused by a clinically relevant pathogen.

Platelet-Dense Granules Worsen Pre-Infection Thrombocytopenia during Gram-Negative Pneumonia-Derived Sepsis

Platelet-dense (δ) granules are important for platelet function. Platelets contribute to host defense and vascular integrity during pneumonia and sepsis, and δ granule products, including adenosine diphosphate (ADP), can influence inflammatory responses. We therefore aimed to study the role of platelet δ granules in the host response during sepsis. Hermansky-Pudlak syndrome (Hps)3coa mice (with reduced δ granule content), mice treated with the platelet ADP receptor inhibitor clopidogrel, and appropriate control mice were infected with the human sepsis pathogen Klebsiella pneumoniae via the airways to induce pneumonia and sepsis. In order to override potential redundancy in platelet functions, we also studied Hps3coa and control mice with moderate antibody-induced thrombocytopenia (10%) prior to infection. We found that sepsis-induced thrombocytopenia tended to be less severe in Hps3coa mice, and was significantly ameliorated in Hps3coa mice with low pre-infection platelet counts. Bacterial growth was similar in Hps3coa and control mice in the presence of normal platelet counts prior to infection, but lower in the lungs of Hps3coa mice with low pre-infection platelet counts. Hps3coa mice had unaltered lung pathology and distant organ injury during pneumosepsis, irrespective of pre-infection platelet counts; lung bleeding did not differ between Hps3coa and control mice. Clopidogrel did not influence any host response parameter. These data suggest that platelet δ granules can play a detrimental role in pneumosepsis by aggravating thrombocytopenia and impairing local antibacterial defense, but that these unfavorable effects only become apparent in the presence of low platelet counts.

Inhibition of the extrinsic or intrinsic coagulation pathway during pneumonia-derived sepsis.

Pneumonia is the most frequent cause of sepsis, and Klebsiella pneumoniae is a common pathogen in pneumonia and sepsis. Infection is associated with activation of the coagulation system. Coagulation can be activated by the extrinsic and intrinsic routes, mediated by factor VII (FVII) and factor XII (FXII), respectively. To determine the role of FVII and FXII in the host response during pneumonia-derived sepsis, mice were treated with specific antisense oligonucleotide (ASO) directed at FVII or FXII for 3 wk before infection with K. pneumoniae via the airways. FVII ASO treatment strongly inhibited hepatic FVII mRNA expression, reduced plasma FVII to ~25% of control, and selectively prolonged the prothrombin time. FXII ASO treatment strongly suppressed hepatic FXII mRNA expression, reduced plasma FXII to ~20% of control, and selectively prolonged the activated partial thromboplastin time. Lungs also expressed FVII mRNA, which was not altered by FVII ASO administration. Very low FXII mRNA levels were detected in lungs, which were not modified by FXII ASO treatment. FVII ASO attenuated systemic activation of coagulation but did not influence fibrin deposition in lung tissue. FVII ASO enhanced bacterial loads in lungs and mitigated sepsis-induced distant organ injury. FXII inhibition did not affect any of the host response parameters measured. These results suggest that partial inhibition of FVII, but not of FXII, modifies the host response to gram-negative pneumonia-derived sepsis.

Role of Peptidylarginine Deiminase 4 in Neutrophil Extracellular Trap Formation and Host Defense during Klebsiella pneumoniae-Induced Pneumonia-Derived Sepsis.

Peptidylarginine deiminase 4 (PAD4) catalyzes citrullination of histones, an important step for neutrophil extracellular trap (NET) formation. We aimed to determine the role of PAD4 during pneumonia. Markers of NET formation were measured in lavage fluid from airways of critically ill patients. NET formation and host defense were studied during pneumonia-derived sepsis caused by Klebsiella pneumoniae in PAD4+/+ and PAD4-/- mice. Patients with pneumosepsis, compared with those with nonpulmonary disease, showed increased citrullinated histone 3 (CitH3) levels in their airways and a trend toward elevated levels of NET markers cell-free DNA and nucleosomes. During murine pneumosepsis, CitH3 levels were increased in the lungs of PAD4+/+ but not of PAD4-/- mice. Combined light and electron microscopy showed NET-like structures surrounding Klebsiella in areas of CitH3 staining in the lung; however, these were also seen in PAD4-/- mice with absent CitH3 lung staining. Moreover, cell-free DNA and nucleosome levels were mostly similar in both groups. Moreover, Klebsiella and LPS could still induce NETosis in PAD4-/- neutrophils. Both groups showed largely similar bacterial growth, lung inflammation, and organ injury. In conclusion, these data argue against a major role for PAD4 in NET formation, host defense, or organ injury during pneumonia-derived sepsis.

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.

The gut microbiota as a modulator of innate immunity during melioidosis.

BackgroundMelioidosis, caused by the Gram-negative bacterium Burkholderia pseudomallei, is an emerging cause of pneumonia-derived sepsis in the tropics. The gut microbiota supports local mucosal immunity and is increasingly recognized as a protective mediator in host defenses against systemic infection. Here, we aimed to characterize the composition and function of the intestinal microbiota during experimental melioidosis.Methodology/Principal findingsC57BL/6 mice were infected intranasally with B. pseudomallei and sacrificed at different time points to assess bacterial loads and inflammation. In selected experiments, the gut microbiota was disrupted with broad-spectrum antibiotics prior to inoculation. Fecal bacterial composition was analyzed by means of IS-pro, a 16S-23S interspacer region-based profiling method. A marked shift in fecal bacterial composition was seen in all mice during systemic B. pseudomallei infection with a strong increase in Proteobacteria and decrease in Actinobacteria, with an increase in bacterial diversity. We found enhanced early dissemination of B. pseudomallei and systemic inflammation during experimental melioidosis in microbiota-disrupted mice compared with controls. Whole-genome transcriptional profiling of the lung identified several genes that were differentially expressed between mice with a normal or disrupted intestinal microbiota. Genes involved in acute phase signaling, including macrophage-related signaling pathways were significantly elevated in microbiota disrupted mice. Compared with controls, alveolar macrophages derived from antibiotic pretreated mice showed a diminished capacity to phagocytose B. pseudomallei. This might in part explain the observed protective effect of the gut microbiota in the host defense against pneumonia-derived melioidosis.Conclusions/SignificanceTaken together, these data identify the gut microbiota as a potential modulator of innate immunity during B. pseudomallei infection.

Thrombin contributes to protective immunity in pneumonia‐derived sepsis via fibrin polymerization and platelet–neutrophil interactions

Background Innate immunity and coagulation are closely linked during sepsis. Their interaction can be detrimental to the outcome because of microvascular failure but can also enhance host defense. The role of thrombin therein has not been fully elucidated. Objective We aimed to investigate the contribution of thrombin to the host response during pneumonia-derived sepsis. Methods Mice treated with the specific thrombin inhibitor dabigatran or control chow were infected with the common human sepsis pathogen Klebsiella (K.) pneumoniae via the airways. In subsequent infection experiments, mice were additionally treated with ancrod to deplete fibrinogen. Ex vivo Klebsiella growth was assessed by incubating human whole blood or specific blood components in various conditions with Klebsiella. Results Thrombin inhibition by dabigatran enhanced bacterial outgrowth and spreading, and accelerated mortality. Thrombin inhibition did not influence neutrophil recruitment to the lung or activation or neutrophil extracellular trap formation. Dabigatran reduced D-dimer formation and fibrin deposition in the lung. Fibrin depletion also enhanced bacterial outgrowth and spreading, and thrombin inhibition had no additional effect. Both thrombin and fibrin polymerization inhibited ex vivo Klebsiella outgrowth in human whole blood, which was neutrophil dependent, and the effect of thrombin required the presence of platelets and platelet protease activated receptor-1. In vivo thrombin inhibition reduced platelet-neutrophil complex formation and endothelial cell activation, but did not prevent sepsis-induced thrombocytopenia or organ damage. Conclusions These results suggest that thrombin plays an important role in protective immunity during pneumonia-derived sepsis by fibrin polymerization and enhancement of platelet-neutrophil interactions.

Coagulation factor XI improves host defence during murine pneumonia-derived sepsis independent of factor XII activation.

Bacterial pneumonia, the most common cause of sepsis, is associated with activation of coagulation. Factor XI (FXI), the key component of the intrinsic pathway, can be activated via factor XII (FXII), part of the contact system, or via thrombin. To determine whether intrinsic coagulation is involved in host defence during pneumonia and whether this is dependent on FXII activation, we infected in parallel wild-type (WT), FXI knockout (KO) and FXII KO mice with two different clinically relevant pathogens, the Gram-positive bacterium Streptococcus pneumoniae and the Gram-negative bacterium Klebsiella pneumoniae, via the airways. FXI deficiency worsened survival and enhanced bacterial outgrowth in both pneumonia models. This was accompanied with enhanced inflammatory responses in FXI KO mice. FXII KO mice were comparable with WT mice in Streptococcus pneumoniae pneumonia. On the contrary, FXII deficiency improved survival and reduced bacterial outgrowth following infection with Klebsiella pneumoniae. In both pneumonia models, local coagulation was not impaired in either FXI KO or FXII KO mice. The capacity to phagocytose bacteria was impaired in FXI KO neutrophils and in human neutrophils where activation of FXI was inhibited. Deficiency for FXII or blocking activation of FXI via FXIIa had no effect on phagocytosis. Taken together, these data suggest that FXI protects against sepsis derived from Streptococcus pneumoniae or Klebsiella pneumoniae pneumonia at least in part by enhancing the phagocytic capacity of neutrophils by a mechanism that is independent of activation via FXIIa.

The role of platelet MyD88 in host response during gram‐negative sepsis

Beside their role in hemostasis, platelets serve as sentinel cells in host defense during infection. In sepsis, platelets have been implicated in both beneficial (antibacterial) and detrimental responses (thrombosis and organ damage). Toll-like receptors and their common adaptor, myeloid differentiation factor 88 (MyD88), are essential for pathogen recognition and protective immunity. Platelets express functional Toll-like receptors and MyD88, which participate in platelet responsiveness to bacterial agonists. Considering the pivotal involvement of platelets and MyD88 in the host response to bacteria, we studied the role of platelet MyD88 in gram-negative sepsis using intravenous and airway infections with the common human sepsis pathogen Klebsiella pneumoniae. Platelet-specific Myd88(-/-) mice were generated by crossing mice with a conditional Myd88 flox allele with mice expressing Cre recombinase controlled by the platelet factor 4 promoter. In a reverse approach, full Myd88(-/-) mice were transfused with wild-type platelets. In both settings, platelet MyD88 did not impact on bacterial growth or dissemination. In addition, platelet MyD88 did not influence hallmark sepsis responses such as thrombocytopenia, coagulation or endothelial activation, or distant organ injury. Platelet MyD88 played no role in lung pathology during pneumonia-derived sepsis. Despite known literature, platelet MyD88-dependent TLR signaling does not contribute to the host response during gram-negative sepsis.