Search

ObjectiveAntimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) represents an enormous risk of failing therapy in hospital-acquired pneumonia. The current study aimed to determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during respiratory infection evoked by hypervirulent antibiotic-susceptible and antibiotic-resistant K. pneumoniae in mice.MethodsC57BL6 mice were inoculated intranasally with hypervirulent K. pneumoniae (K2:O1) which was either antibiotic-susceptible or multi-drug resistant. Six hours after infection, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally. Mice were sacrificed 24 hours after infection. Samples were analyzed for bacterial loads and for inflammatory and coagulation markers.ResultsFlagellin therapy induced neutrophil influx in the lung during antibiotic-treated pneumonia evoked by either antibiotic-susceptible or -resistant K. pneumoniae. The pulmonary neutrophil response was matched by elevated levels of neutrophil-attracting chemokines, neutrophil degranulation products, and local coagulation activation. The combined therapy of effective antibiotics and flagellin did not impact K. pneumoniae outgrowth in the lung, but decreased bacterial counts in distant organs. Neutrophil depletion abrogated the flagellin-mediated effect on bacterial dissemination and local coagulation responses.ConclusionTopical flagellin administration as an adjunctive to antibiotic treatment augments neutrophil responses during pneumonia evoked by MDR-K. pneumoniae, thereby reducing bacterial dissemination to distant organs.

Introduction: Oral anticoagulant therapy comes at the cost of a significant bleeding risk. However, it is hard to predict which patients are at risk of major bleeding. Previously we found associations of Calibrated Automated Thrombinography (CAT) parameters obtained in the presence of TIX-5 (an inhibitor of the FV activation by FXa), and plasma levels of TFPIα, γ’-fibrinogen and soluble thrombomodulin with major bleeding in the BLEEDS cohort, a cohort especially powered to find new biomarkers of major bleeding during VKA therapy. Methods: To determine and compare the predictive capability for major bleeding in the BLEEDS cohort of the above biomarkers, also in a combined model with clinical risk factors, we performed Univariable Prentice-weighted Cox regression analyses and Bayesian variable selection. Results: The highest predictive value among the laboratory measures were found for thrombin generation lagtime in the presence of TIX-5 (TIX-5 lagtime per 25% increase, hazard ratio (HR) 1.11, 95%CI 1.04–1.18, p=0.001) and full-length tissue factor pathway inhibitor (TFPIα) (per 25% increase HR 1.12, 95%CI 1.03–1.21, p=0.008), which remained significant after correction for multiple testing, and independently associated with major bleeding after Bayesian variable selection. Only the addition of TIX-5 lagtime to the clinical risk factors improved prediction of major bleeding significantly (p

The anti-inflammatory interleukin-1 receptor associated kinase-M (IRAK-M) is a negative regulator of MyD88/IRAK-4/IRAK-1 signaling. However, IRAK-M has also been reported to activate NF-κB through the MyD88/IRAK-4/IRAK-M myddosome in a MEKK-3 dependent manner. Here we provide support that IRAK-M uses three surfaces of its Death Domain (DD) to activate NF-κB downstream of MyD88/IRAK-4/IRAK-M. Surface 1, with central residue Trp74, binds to MyD88/IRAK-4. Surface 2, with central Lys60, associates with other IRAK-M DDs to form an IRAK-M homotetramer under the MyD88/IRAK-4 scaffold. Surface 3; with central residue Arg97 is located on the opposite side of Trp74 in the IRAK-M DD tetramer, lacks any interaction points with the MyD88/IRAK-4 complex. Although the IRAK-M DD residue Arg97 is not directly involved in the association with MyD88/IRAK-4, Arg97 was responsible for 50% of the NF-κB activation though the MyD88/IRAK-4/IRAK-M myddosome. Arg97 was also found to be pivotal for IRAK-M’s interaction with IRAK-1, and important for IRAK-M’s interaction with TRAF6. Residue Arg97 was responsible for 50% of the NF-κB generated by MyD88/IRAK-4/IRAK-M myddosome in IRAK-1/MEKK3 double knockout cells. By structural modeling we found that the IRAK-M tetramer surface around Arg97 has excellent properties that allow formation of an IRAK-M homo-octamer. This model explains why mutation of Arg97 results in an IRAK-M molecule with increased inhibitory properties: it still binds to myddosome, competing with myddosome IRAK-1 binding, while resulting in less NF-κB formation. The findings further identify the structure-function properties of IRAK-M, which is a potential therapeutic target in inflammatory disease.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

Monocytes are key players in innate immunity, with their ability to regulate inflammatory responses and combat invading pathogens. There is a growing body of evidence indicating that long non-coding RNA (lncRNA) participate in various cellular biological processes, including the innate immune response. The immunoregulatory properties of numerous lncRNAs discovered in monocytes remain largely unexplored. Here, by RNA sequencing, we identified a lncRNA JHDM1D-AS1, which was upregulated in blood monocytes obtained from patients with sepsis relative to healthy controls. JHDM1D-AS1 expression was induced in primary human monocytes exposed to Toll-like receptor ligands, such as lipopolysaccharide (LPS), or bacteria. The inducibility of JHDM1D-AS1 expression in monocytes depended, at least in part, on nuclear factor–κB activation. JHDM1D-AS1 knockdown experiments in human monocyte-derived macrophages revealed significantly enhanced expression of inflammatory mediators, before and after exposure to LPS, relative to control cells. Specifically, genes involved in inflammatory responses were upregulated (e.g., CXCL2, CXCL8, IL1RN, TREM1, TNF, and IL6), whereas genes involved in anti-inflammatory pathways were downregulated (e.g., SOCS1 and IL10RA). JHDM1D-AS1 overexpression in a pro-monocytic cell line revealed diminished pro-inflammatory responses subsequent to LPS challenge. Collectively, these findings identify JHDM1D-AS1 as a potential anti-inflammatory mediator induced in response to inflammatory stimuli.

Our previous work identified human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1) as a putative driver of LPS-induced NF-κB signaling in humans in vivo. While HIVEP1 is known to interact with NF-ĸB binding DNA motifs, its function in mammalian cells is unknown. We report increased HIVEP1 mRNA expression in monocytes from patients with sepsis and monocytes stimulated by Toll-like receptor agonists and bacteria. In complementary overexpression and gene deletion experiments HIVEP1 was shown to inhibit NF-ĸB activity and induction of NF-ĸB responsive genes. RNA sequencing demonstrated profound transcriptomic changes in HIVEP1 deficient monocytic cells and transcription factor binding site analysis showed enrichment for κB site regions. HIVEP1 bound to the promoter regions of NF-ĸB responsive genes. Inhibition of cytokine production by HIVEP1 was confirmed in LPS-stimulated murine Hivep1-/- macrophages and HIVEP1 knockdown zebrafish exposed to the common sepsis pathogen Streptococcus pneumoniae. These results identify HIVEP1 as a negative regulator of NF-κB in monocytes/macrophages that inhibits proinflammatory reactions in response to bacterial agonists in vitro and in vivo.

Bruton’s tyrosine kinase (Btk) is a cytoplasmic kinase expressed in B cells and myeloid cells. It is essential for B cell development and natural antibody-mediated host defense against bacteria in humans and mice, but little is known about the role of Btk in innate host defense in vivo. Previous studies have indicated that lack of (natural) antibodies is paramount for impaired host defense against Streptococcus (S.) pneumoniae in patients and mice with a deficiency in functional Btk. In the present study, we re-examined the role of Btk in B cells and myeloid cells during pneumococcal pneumonia and sepsis in mice. The antibacterial defense of Btk-/- mice was severely impaired during pneumococcal pneumosepsis and restoration of natural antibody production in Btk-/- mice by transgenic expression of Btk specifically in B cells did not suffice to protect against infection. Btk-/- mice with reinforced Btk expression in MhcII+ cells, including B cells, dendritic cells and macrophages, showed improved antibacterial defense as compared to Btk-/- mice. Bacterial outgrowth in Lysmcre-Btkfl/Y mice was unaltered despite a reduced capacity of Btk-deficient alveolar macrophages to respond to pneumococci. Mrp8cre-Btkfl/Y mice with a neutrophil specific paucity in Btk expression, however, demonstrated impaired antibacterial defense. Neutrophils of Mrp8cre-Btkfl/Y mice displayed reduced release of granule content after pulmonary installation of lipoteichoic acid, a gram-positive bacterial cell wall component relevant for pneumococci. Moreover, Btk deficient neutrophils showed impaired degranulation and phagocytosis upon incubation with pneumococci ex vivo. Taken together, the results of our study indicate that besides regulating B cell-mediated immunity, Btk is critical for regulation of myeloid cell-mediated, and particularly neutrophil-mediated, innate host defense against S. pneumoniae in vivo.

Abstract Background Complement factor C5 can either aggravate or attenuate the T-helper type 2 (TH2) immune response and airway hyperresponsiveness (AHR) in murine models of allergic asthma. The effect of C5 during the effector phase of allergen-induced asthma is ill-defined. Objectives We aimed to determine the effect of C5 blockade during the effector phase on the pulmonary TH2 response and AHR in a house dust mite (HDM) driven murine asthma model. Methods BALB/c mice were sensitized and challenged repeatedly with HDM via the airways to induce allergic lung inflammation. Sensitized mice received twice weekly injections with a blocking anti-C5 or control antibody 24 h before the first challenge. Results HDM challenge in sensitized mice resulted in elevated C5a levels in bronchoalveolar lavage fluid. Anti-C5 administered to sensitized mice prior to the first HDM challenge prevented this rise in C5a, but did not influence the influx of eosinophils or neutrophils. While anti-C5 did not impact the recruitment of CD4 T cells upon HDM challenge, it reduced the proportion of TH2 cells recruited to the airways, attenuated IL-4 release by regional lymph nodes restimulated with HDM ex vivo and mitigated the plasma IgE response. Anti-C5 did not affect innate lymphoid cell (ILC) proliferation or group 2 ILC (ILC2) differentiation. Anti-C5 attenuated HDM induced AHR in the absence of an effect on lung histopathology, mucus production or vascular leak. Conclusions Generation of C5a during the effector phase of HDM induced allergic lung inflammation contributes to TH2 cell differentiation and AHR without impacting ILC2 cells.

Platelets and Toll-like receptor (TLR) signalling play a role in the immune response during sepsis. Although preclinical knowledge about the role of platelet TLR signalling is increasing, data during human sepsis are less abundant. Moreover, controversy remains about the effect of the TLR4 agonist lipopolysaccharide (LPS) on platelet activation. We therefore assessed platelet TLR expression during human and murine sepsis. Moreover, we investigated the effect of TLR4 signalling on platelet activation and TLR expression. Platelets from healthy controls stimulated with LPS did not show classical platelet activation (P-selectin, CD63 and phosphatidylserine expression), potentiation of subthreshold agonist stimulation nor platelet-leukocyte complex formation. LPS stimulation however did increase maximal mitochondrial respiration in a TLR4-dependent manner. Platelet stimulation with LPS did not alter TLR expression. Platelet stimulation with thrombin receptor activating peptide increased TLR5 and TLR9, but not TLR2 or TLR4 expression. Platelets from patients with sepsis and mice with experimental sepsis showed platelet activation, but unaltered TLR expression. These results indicate that sepsis-induced platelet activation is not associated with altered platelet TLR expression and, although platelets are responsive to LPS, stimulation of platelet TLR4 does not result in classical platelet activation.

C1 esterase inhibitor (C1-INH) can inhibit multiple pathways (complement, contact-kinin, coagulation, and fibrinolysis) that are all implicated in the pathophysiology of asthma. We explored the effect of human plasma-derived C1-INH on allergic lung inflammation in a house dust mite (HDM) induced asthma mouse model by daily administration of C1-INH (15 U) during the challenge phase. NaCl and HDM exposed mice had comparable plasma C1-INH levels, while bronchoalveolar lavage fluid (BALF) levels were increased in HDM exposed mice coinciding with slightly reduced activation of complement (C5a). C1-INH treatment reduced Th2 response and enhanced HDM-specific IgG1. Influx of eosinophils in BALF or lung, pulmonary damage, mucus production, procoagulant response or plasma leakage in BALF was similar in both groups. In conclusion, C1-INH dampens Th2 responses during HDM induced allergic lung inflammation.

Borrelia burgdorferi is transmitted into the skin of the host where it encounters and interacts with two dendritic cell (DC) subsets; Langerhans cells (LCs) and dermal DCs (DDCs). These cells recognize pathogens via pattern recognition receptors, mature and migrate out of the skin into draining lymph nodes, where they orchestrate adaptive immune responses. In order to investigate the response of skin DCs during the early immunopathogenesis of Lyme borreliosis, we injected B. burgdorferi intradermally into full-thickness human skin and studied the migration of DCs out of the skin, the activation profile and phenotype of migrated cells. We found a significant increase in the migration of LCs and DDCs in response to B. burgdorferi. Notably, migration was prevented by blocking TLR2. DCs migrated from skin inoculated with higher numbers of spirochetes expressed significantly higher levels of CD83 and produced pro-inflammatory cytokines. No difference was observed in the expression of HLA-DR, CD86, CD38, or CCR7. To conclude, we have established an ex vivo human skin model to study DC-B. burgdorferi interactions. Using this model, we have demonstrated that B. burgdorferi-induced DC migration is mediated by TLR2. Our findings underscore the utility of this model as a valuable tool to study immunity to spirochetal infections.

Streptococcus (S.) pneumoniae strains vary considerably in their ability to cause invasive disease in humans, which is at least in part determined by the capsular serotype. Platelets have been implicated as sentinel cells in the circulation for host defence. One of their utensils for this function is the expression of Toll-like receptors (TLRs). We here aimed to investigate platelet response to S. pneumoniae and a role for TLRs herein. Platelets were stimulated using four serotypes of S. pneumonia including an unencapsulated mutant strain. In vitro aggregation and flow cytometry assays were performed using blood of healthy volunteers, or blood of TLR knock out and WT mice. For in vivo pneumonia experiments, platelet specific Myd88 knockout (Plt-Myd88-/-) mice were used. We found that platelet aggregation was induced by unencapsulated S. pneumoniae only. Whole blood incubation with all S. pneumoniae serotypes tested resulted in platelet degranulation and platelet-leukocyte complex formation. Platelet activation was TLR independent, as responses were not inhibited by TLR blocking antibodies, not induced by TLR agonists and were equally induced in wild-type and Tlr2-/-, Tlr4-/-, Tlr2/4-/-, Tlr9-/- and Myd88-/- blood. Plt-Myd88-/- and control mice displayed no differences in bacterial clearance or immune response to pneumonia by unencapsulated S. pneumoniae. In conclusion, S. pneumoniae activates platelets through a TLR-independent mechanism that is impeded by the bacterial capsule. Additionally, platelet MyD88-dependent TLR signalling is not involved in host defence to unencapsulated S. pneumoniae in vivo.

Klebsiella species is the second most commonly isolated gram-negative organism in sepsis and a frequent causative pathogen in pneumonia. The receptor for advanced glycation end products (RAGE) is expressed on different cell types and plays a key role in diverse inflammatory responses. We here aimed to investigate the role of RAGE in the host response to Klebsiella (K.) pneumoniae pneumonia and intransally inoculated rage gene deficient (RAGE-/-) and normal wild-type (Wt) mice with K. pneumoniae. Klebsiella pneumonia resulted in an increased pulmonary expression of RAGE. Furthermore, the high-affinity RAGE ligand high mobility group box-1 was upregulated during K. pneumoniae pneumonia. RAGE deficiency impaired host defense as reflected by a worsened survival, increased bacterial outgrowth and dissemination in RAGE-/- mice. RAGE-/- neutrophils showed a diminished phagocytosing capacity of live K. pneumoniae in vitro. Relative to Wt mice, RAGE-/- mice demonstrated similar lung inflammation, and slightly elevated-if any-cytokine and chemokine levels and unchanged hepatocellular injury. In addition, RAGE-/- mice displayed an unaltered response to intranasally instilled Klebsiella lipopolysaccharide (LPS) with respect to pulmonary cell recruitment and local release of cytokines and chemokines. These data suggest that (endogenous) RAGE protects against K. pneumoniae pneumonia. Also, they demonstrate that RAGE contributes to an effective antibacterial defense during K. pneumoniae pneumonia, at least partly via its participation in the phagocytic properties of professional granulocytes. Additionally, our results indicate that RAGE is not essential for the induction of a local and systemic inflammatory response to either intact Klebsiella or Klebsiella LPS.

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD) 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2-/-) and wild-type (Wt) alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39), an isogenic capsule locus deletion mutant (D39Δcps) or serotype 3 S. pneumoniae (6303) via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.

Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88(-/-) mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88(-/-) mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88(-/-) mice showed 105-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.

The Gram-negative bacterium Burkholderia pseudomallei causes melioidosis and is a CDC category B bioterrorism agent. Toll-like receptor (TLR)-2 impairs host defense during pulmonary B.pseudomallei infection while TLR4 only has limited impact. We investigated the role of TLRs in B.pseudomallei-lipopolysaccharide (LPS) induced inflammation. Purified B.pseudomallei-LPS activated only TLR2-transfected-HEK-cells during short stimulation but both HEK-TLR2 and HEK-TLR4-cells after 24 h. In human blood, an additive effect of TLR2 on TLR4-mediated signalling induced by B.pseudomallei-LPS was observed. In contrast, murine peritoneal macrophages recognized B.pseudomallei-LPS solely through TLR4. Intranasal inoculation of B.pseudomallei-LPS showed that both TLR4-knockout(-/-) and TLR2x4-/-, but not TLR2-/- mice, displayed diminished cytokine responses and neutrophil influx compared to wild-type controls. These data suggest that B.pseudomallei-LPS signalling occurs solely through murine TLR4, while in human models TLR2 plays an additional role, highlighting important differences between specificity of human and murine models that may have important consequences for B.pseudomallei-LPS sensing by TLRs and subsequent susceptibility to melioidosis.

Pulmonary coagulopathy is intrinsic to pulmonary injury including pneumonia. Anticoagulant strategies could benefit patients with pneumonia, but systemic administration of anticoagulant agents may lead to suboptimal local levels and may cause systemic hemorrhage. We hypothesized nebulization to provide a safer and more effective route for local administration of anticoagulants. Therefore, we aimed to examine feasibility and safety of nebulization of recombinant human tissue factor pathway inhibitor (rh-TFPI) in a well-established rat model of Streptococcus (S.) pneumoniae pneumonia. Thirty minutes before and every 6 hours after intratracheal instillation of S. pneumonia causing pneumonia, rats were subjected to local treatment with rh-TFPI or placebo, and sacrificed after 42 hours. Pneumonia was associated with local as well as systemic activation of coagulation. Nebulization of rh-TFPI resulted in high levels of rh-TFPI in bronchoalveolar lavage fluid, which was accompanied by an attenuation of pulmonary coagulation. Systemic rh-TFPI levels remained undetectable, and systemic TFPI activity and systemic coagulation were not affected. Histopathology revealed no bleeding in the lungs. We conclude that nebulization of rh-TFPI seems feasible and safe; local anticoagulant treatment with rh-TFPI attenuates pulmonary coagulation, while not affecting systemic coagulation in a rat model of S. pneumoniae pneumonia.

BACKGROUND: During severe (pneumo)sepsis inflammatory and coagulation pathways become activated as part of the host immune response. Thrombomodulin (TM) is involved in a range of host defense mechanisms during infection and plays a pivotal role in activation of protein C (PC) into active protein C (APC). APC has both anticoagulant and anti-inflammatory properties. In this study we investigated the effects of impaired TM-mediated APC generation during melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia caused by Burkholderia (B.) pseudomallei. METHODOLOGY/PRINCIPAL FINDINGS: (WT) mice and mice with an impaired capacity to activate protein C due to a point mutation in their Thbd gene (TMpro/pro mice) were intranasally infected with B. pseudomallei and sacrificed after 24, 48 or 72 hours for analyses. Additionally, survival studies were performed. When compared to WT mice, TMpro/pro mice displayed a worse survival upon infection with B. pseudomallei, accompanied by increased coagulation activation, enhanced lung neutrophil influx and bronchoalveolar inflammation at late time points, together with increased hepatocellular injury. The TMpro/pro mutation had limited if any impact on bacterial growth and dissemination. CONCLUSION/SIGNIFICANCE: TM-mediated protein C activation contributes to protective immunity after infection with B. pseudomallei. These results add to a better understanding of the regulation of the inflammatory and procoagulant response during severe Gram-negative (pneumo)sepsis.

TNFα has been implicated in the pathogenesis of various inflammatory diseases. Different strategies to inhibit TNFα in patients with sepsis and chronic inflammatory conditions have shown contrasting outcomes. Although TNFα inhibitors are widely used in clinical practice, the impact of TNFα antagonism on white blood cell gene expression profiles during acute inflammation in humans in vivo has not been assessed. We here leveraged the established model of human endotoxemia to examine the effect of the TNFα antagonist, etanercept, on the genome-wide transcriptional responses in circulating leukocytes induced by intravenous LPS administration in male subjects. Etanercept pre-treatment resulted in a markedly dampened transcriptional response to LPS. Gene co-expression network analysis revealed this LPS-induced transcriptome can be categorized as TNFα responsive and non-responsive modules. Highly significant TNFα responsive modules include NF-kB signaling, antiviral responses and T-cell mediated responses. Within these TNFα responsive modules we delineate fundamental genes involved in epigenetic modifications, transcriptional initiation and elongation. Thus, we provide comprehensive information about molecular pathways that might be targeted by therapeutic interventions that seek to inhibit TNFα activity during human inflammatory diseases.

Streptococcus (S.) pneumoniae is a common causative pathogen in pneumonia. Serine protease orthologs expressed by a variety of bacteria have been found of importance for virulence. Previous studies have identified two serine proteases in S. pneumoniae, HtrA (high-temperature requirement A) and PrtA (cell wall-associated serine protease A), that contributed to virulence in models of pneumonia and intraperitoneal infection respectively. We here sought to identify additional S. pneumoniae serine proteases and determine their role in virulence. The S. pneumoniae D39 genome contains five putative serine proteases, of which HtrA, Subtilase Family Protein (SFP) and PrtA were selected for insertional mutagenesis because they are predicted to be secreted and surface exposed. Mutant D39 strains lacking serine proteases were constructed by in-frame insertion deletion mutagenesis. Pneumonia was induced by intranasal infection of mice with wild-type or mutant D39. After high dose infection, only D39ΔhtrA showed reduced virulence, as reflected by strongly reduced bacterial loads, diminished dissemination and decreased lung inflammation. D39ΔprtA induced significantly less lung inflammation together with smaller infiltrated lung surface, but without influencing bacterial loads. After low dose infection, D39ΔhtrA again showed strongly reduced bacterial loads; notably, pneumococcal burdens were also modestly lower in lungs after infection with D39Δsfp. These data confirm the important role for HtrA in S. pneumoniae virulence. PrtA contributes to lung damage in high dose pneumonia; it does not however contribute to bacterial outgrowth in pneumococcal pneumonia. SFP may facilitate S. pneumoniae growth after low dose infection.

The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia.Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR(-/-)). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR(-/-) mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma.Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis.

Interleukin-1 receptor like 1 (ST2) is a negative regulator of Toll-like receptor (TLR) signaling. TLRs are important for host defense during respiratory tract infections by both influenza and Streptococcus (S.) pneumoniae. Enhanced susceptibility to pneumococcal pneumonia is an important complication following influenza virus infection. We here sought to determine the role of ST2 in primary influenza A infection and secondary pneumococcal pneumonia. ST2 knockout (st2(-/-)) and wild-type (WT) mice were intranasally infected with influenza A virus; in some experiments mice were infected 2 weeks later with S. pneumoniae. Both mouse strains cleared the virus similarly during the first 14 days of influenza infection and had recovered their weights equally at day 14. Overall st2(-/-) mice tended to have a stronger pulmonary inflammatory response upon infection with influenza; especially 14 days after infection modest but statistically significant elevations were seen in lung IL-6, IL-1β, KC, IL-10, and IL-33 concentrations and myeloperoxidase levels, indicative of enhanced neutrophil activity. Interestingly, bacterial lung loads were higher in st2(-/-) mice during the later stages of secondary pneumococcal pneumonia, which was associated with relatively increased lung IFN-γ levels. ST2 deficiency did not impact on gross lung pathology in either influenza or secondary S. pneumoniae pneumonia. These data show that ST2 plays a limited anti-inflammatory role during both primary influenza and postinfluenza pneumococcal pneumonia.

Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Myeloid related protein 8 (MRP8, S100A8) and MRP14 (S100A9) are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released upon cell stress stimuli. MRP8/14 reportedly exerts antimicrobial activity, but in acute fulminant sepsis models MRP8/14 has been found to contribute to organ damage and death. We here determined the role of MRP8/14 in K. pneumoniae sepsis originating from the lungs, using an established model characterized by gradual growth of bacteria with subsequent dissemination. Infection resulted in gradually increasing MRP8/14 levels in lungs and plasma. Mrp14 deficient (mrp14(-/-)) mice, unable to form MRP8/14 heterodimers, showed enhanced bacterial dissemination accompanied by increased organ damage and a reduced survival. Mrp14(-/-) macrophages were reduced in their capacity to phagocytose Klebsiella. In addition, recombinant MRP8/14 heterodimers, but not MRP8 or MRP14 alone, prevented growth of Klebsiella in vitro through chelation of divalent cations. Neutrophil extracellular traps (NETs) prepared from wildtype but not from mrp14(-/-) neutrophils inhibited Klebsiella growth; in accordance, the capacity of human NETs to kill Klebsiella was strongly impaired by an anti-MRP14 antibody or the addition of zinc. These results identify MRP8/14 as key player in protective innate immunity during Klebsiella pneumonia.

BACKGROUND: Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires. CONCLUSIONS/SIGNIFICANCE: Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.

BACKGROUND: Recognition of lipopolysaccharide (LPS) is required for effective defense against invading gram-negative bacteria. Recently, in vitro studies revealed that CD14 is required for activation of the myeloid differentiation factor (MyD)88-dependent Toll-like receptor (TLR)4 signaling pathway by smooth (S)-LPS, but not by rough (R)-LPS. The present study investigated the role of CD14 in induction of lung inflammation in mice by these different LPS chemotypes. METHODOLOGY/RESULTS: Neutrophil accumulation and tumor necrosis factor (TNF) release in bronchoalveolar lavage fluid were determined 6 hours after intranasal treatment of wild type (WT) and CD14 knock-out (KO) mice with different doses S-LPS or R-LPS. The contribution of CD14 to lung inflammation induced by S-LPS or R-LPS depended on the LPS dose. At low doses, S-LPS and R-LPS induced neutrophil influx in a CD14-dependent manner. Low dose S-LPS-induced cytokine release also depended on CD14. Strikingly, neutrophil influx and TNF release induced by high dose S-LPS or R-LPS was diminished in the presence of CD14. Intranasal administration of sCD14 to CD14 KO mice treated with S-LPS partially reversed the inflammatory response to the response observed in WT mice. CONCLUSIONS: In conclusion, CD14 modulates effects of both S-LPS and R-LPS within the lung in a similar way. Except for R-LPS-induced TNF release, S-LPS and R-LPS at low dose induced acute lung inflammation in a CD14-dependent manner, while the inflammatory response triggered by high dose S-LPS or R-LPS was diminished by CD14.

Severe leptospirosis features bleeding and multi-organ failure, leading to shock and death. Currently it is assumed that both exaggerated inflammation and immune suppression contribute to mortality in sepsis. Indeed, several proinflammatory cytokines are reported to be induced during leptospirosis. Toll-like receptors, which play an important role in the initiation of an innate immune response, are inhibited by negative regulators including the membrane-bound ST2 (mST2) receptor. Soluble ST2 (sST2) has been implicated to inhibit signaling through mST2. The aim of this study was to determine the extent of sST2 and (pro-) inflammatory cytokine release in patients with severe leptospirosis.In an observational study, 68 consecutive cases of severe leptospirosis were included. Soluble ST2 and cytokines (TNF-alpha, IL-1beta, IL-6, IL-8, and IL-10) were repeatedly measured. To determine whether blood cells are a source of sST2 during infection, we undertook an in vitro experiment: human whole blood and peripheral blood mononuclear cells (PBMC) were stimulated with viable pathogenic Leptospira. All patients showed elevated sST2, IL-6, IL-8, and IL-10 levels on admission. Admission sST2 levels correlated with IL-6, IL-8, and IL-10. Thirty-four patients (50%) showed clinical bleeding. Soluble ST2 levels were significantly associated with bleeding overall (OR 2.0; 95%CI: 1.2-3.6) and severe bleeding (OR 5.1; 95%CI: 1.1-23.8). This association was unique, since none of the cytokines showed this correlation. Moreover, sST2 was associated with mortality (OR 2.4; 95%CI: 1.0-5.8). When either whole blood or isolated PBMCs were stimulated with Leptospira in vitro, no sST2 production could be detected.Patients with severe leptospirosis demonstrated elevated plasma sST2 levels. Soluble ST2 levels were associated with bleeding and mortality. In vitro experiments showed that (white) blood cells are probably not the source. In this regard, sST2 could be an indicative marker for tissue damage in patients suffering from severe leptospirosis.

The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity) and PAI-1 (high fibrinolytic activity) knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1beta mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.