Post-resolution macrophages shape long-term tissue immunity and integrity in a mouse model of pneumococcal pneumonia.

Resolving inflammation is thought to return the affected tissue back to homoeostasis but recent evidence supports a non-linear model of resolution involving a phase of prolonged immune activity. Here we show that within days following resolution of Streptococcus pneumoniae-triggered lung inflammation, there is an influx of antigen specific lymphocytes with a memory and tissue-resident phenotype as well as macrophages bearing alveolar or interstitial phenotype. The transcriptome of these macrophages shows enrichment of genes associated with prostaglandin biosynthesis and genes that drive T cell chemotaxis and differentiation. Therapeutic depletion of post-resolution macrophages, inhibition of prostaglandin E2 (PGE2) synthesis or treatment with an EP4 antagonist, MF498, reduce numbers of lung CD4+/CD44+/CD62L+ and CD4+/CD44+/CD62L-/CD27+ T cells as well as their expression of the α-integrin, CD103. The T cells fail to reappear and reactivate upon secondary challenge for up to six weeks following primary infection. Concomitantly, EP4 antagonism through MF498 causes accumulation of lung macrophages and marked tissue fibrosis. Our study thus shows that PGE2 signalling, predominantly via EP4, plays an important role during the second wave of immune activity following resolution of inflammation. This secondary immune activation drives local tissue-resident T cell development while limiting tissue injury.

CETP inhibition enhances monocyte activation and bacterial clearance and reduces streptococcus pneumonia-associated mortality in mice.

Sepsis is a leading cause of mortality worldwide, and pneumonia is the most common cause of sepsis in humans. Low levels of high-density lipoprotein cholesterol (HDL-C) levels are associated with an increased risk of death from sepsis, and increasing levels of HDL-C by inhibition of cholesteryl ester transfer protein (CETP) decreases mortality from intraabdominal polymicrobial sepsis in APOE*3-Leiden.CETP mice. Here, we show that treatment with the CETP inhibitor (CETPi) anacetrapib reduced mortality from Streptococcus pneumoniae-induced sepsis in APOE*3-Leiden.CETP and APOA1.CETP mice. Mechanistically, CETP inhibition reduced the host proinflammatory response via attenuation of proinflammatory cytokine transcription and release. This effect was dependent on the presence of HDL, leading to attenuation of immune-mediated organ damage. In addition, CETP inhibition promoted monocyte activation in the blood prior to the onset of sepsis, resulting in accelerated macrophage recruitment to the lung and liver. In vitro experiments demonstrated that CETP inhibition significantly promoted the activation of proinflammatory signaling in peripheral blood mononuclear cells and THP1 cells in the absence of HDL; this may represent a mechanism responsible for improved bacterial clearance during sepsis. These findings provide evidence that CETP inhibition represents a potential approach to reduce mortality from pneumosepsis.

Degradation of EGFR on lung epithelial cells by neutrophil elastase contributes to the aggravation of pneumococcal pneumonia.

Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia.

TNFR2+ regulatory T cells protect against bacteremic pneumococcal pneumonia by suppressing IL-17A-producing γδ T cells in the lung.

Streptococcus pneumoniae is a pathogen of global morbidity and mortality. Pneumococcal pneumonia can lead to systemic infections associated with high rates of mortality. We find that, upon pneumococcal infection, pulmonary Treg cells are activated and have upregulated TNFR2 expression. TNFR2-deficient mice have compromised Treg cell responses and highly activated IL-17A-producing γδ T cell (γδT17) responses, resulting in significantly enhanced neutrophil infiltration, tissue damage, and rapid development of bacteremia, mirroring responses in Treg cell-depleted mice. Deletion of total Treg cells predominantly activate IFNγ-T cell responses, whereas adoptive transfer of TNFR2+ Treg cells specifically suppress the γδT17 response, suggesting a targeted control of γδT17 activation by TNFR2+ Treg cells. Blocking IL-17A at early stage of infection significantly reduces bacterial blood dissemination and improves survival in TNFR2-deficient mice. Our results demonstrate that TNFR2 is critical for Treg cell-mediated regulation of pulmonary γδT17-neutrophil axis, with impaired TNFR2+ Treg cell responses increasing susceptibility to disease.

Protective role of Cav-1 in pneumolysin-induced endothelial barrier dysfunction.

Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling.

IL-6 Prevents Lung Macrophage Death and Lung Inflammation Injury by Inhibiting GSDME- and GSDMD-Mediated Pyroptosis during Pneumococcal Pneumosepsis.

Streptococcus pneumoniae is a leading bacterial cause of a wide range of infections, and pneumococcal pneumosepsis causes high mortality in hosts infected with antibiotic-resistant strains and those who cannot resolve ongoing inflammation. The factors which influence the development and outcome of pneumosepsis are currently unclear. IL-6 is critical for maintaining immune homeostasis, and we determined that this cytokine is also essential for resisting pneumosepsis, as it inhibits macrophage pyroptosis and pyroptosis-related inflammation injury in the lung. IL-6 affected infection outcomes in mice and exerted a protective role, primarily via macrophages. We further found that IL-6 deficiency led to increased lung macrophage death and aggravated lung inflammation, and that exogenous administration of IL-6 protein could decrease macrophage death and alleviate lung tissue inflammation. IL-6 also protected Streptococcus pneumoniae-induced lung macrophage death and lung inflammation injury by inhibiting gasdermin E (GSDME)- and gasdermin D (GSDMD)-mediated pyroptosis. Together, these data reveal a novel mechanism for the development of pneumosepsis and the critical protective role of IL-6. These findings may assist in the early identification and treatment of pneumococcal pneumosepsis. IMPORTANCE Pneumococcal pneumonia has been a significant cause of morbidity and mortality throughout human history. Failing to control pneumococcal pneumonia and resolve ongoing inflammation in a host can cause sepsis, namely pneumococcal pneumosepsis, and death ensues. Few theories have suggested an optimally therapeutic option for this infectious disease. The interleukin-6 (IL-6, a cytokine featuring pleiotropic activity) theory, proposed here, implies that IL-6 acts as a protector against pneumococcal pneumosepsis. IL-6 prevents lung macrophage death and lung inflammation injury by inhibiting a caspase-3-GSDME-mediated switch from apoptosis to pyroptosis and inhibiting caspase-1-GSDMD-mediated classic pyroptosis during pneumococcal pneumosepsis. Thus, IL-6 is an important determinant for controlling bacterial invasion and a homeostatic coordinator of pneumococcal pneumosepsis. This study clarifies a novel mechanism of occurrence and development of pneumonia and secondary sepsis following a Streptococcus pneumoniae infection. It is important for the early identification and treatment of pneumococcal pneumosepsis.

ZIP8-Mediated Intestinal Dysbiosis Impairs Pulmonary Host Defense against Bacterial Pneumonia.

Pneumococcal pneumonia is a leading cause of morbidity and mortality worldwide. An increased susceptibility is due, in part, to compromised immune function. Zinc is required for proper immune function, and an insufficient dietary intake increases the risk of pneumonia. Our group was the first to reveal that the Zn transporter, ZIP8, is required for host defense. Furthermore, the gut microbiota that is essential for lung immunity is adversely impacted by a commonly occurring defective ZIP8 allele in humans. Taken together, we hypothesized that loss of the ZIP8 function would lead to intestinal dysbiosis and impaired host defense against pneumonia. To test this, we utilized a novel myeloid-specific Zip8KO mouse model in our studies. The comparison of the cecal microbial composition of wild-type and Zip8KO mice revealed significant differences in microbial community structure. Most strikingly, upon a S. pneumoniae lung infection, mice recolonized with Zip8KO-derived microbiota exhibited an increase in weight loss, bacterial dissemination, and lung inflammation compared to mice recolonized with WT microbiota. For the first time, we reveal the critical role of myeloid-specific ZIP8 on the maintenance of the gut microbiome structure, and that loss of ZIP8 leads to intestinal dysbiosis and impaired host defense in the lung. Given the high incidence of dietary Zn deficiency and the ZIP8 variant allele in the human population, additional investigation is warranted to improve surveillance and treatment strategies.

Adverse Mechanical Ventilation and Pneumococcal Pneumonia Induce Immune and Mitochondrial Dysfunctions Mitigated by Mesenchymal Stem Cells in Rabbits.

BACKGROUND: Mechanical ventilation for pneumonia may contribute to lung injury due to factors that include mitochondrial dysfunction, and mesenchymal stem cells may attenuate injury. This study hypothesized that mechanical ventilation induces immune and mitochondrial dysfunction, with or without pneumococcal pneumonia, that could be mitigated by mesenchymal stem cells alone or combined with antibiotics. METHODS: Male rabbits underwent protective mechanical ventilation (8 ml/kg tidal volume, 5 cm H2O end-expiratory pressure) or adverse mechanical ventilation (20 ml/kg tidal-volume, zero end-expiratory pressure) or were allowed to breathe spontaneously. The same settings were then repeated during pneumococcal pneumonia. Finally, infected animals during adverse mechanical ventilation received human umbilical cord-derived mesenchymal stem cells (3 × 106/kg, intravenous) and/or ceftaroline (20 mg/kg, intramuscular) or sodium chloride, 4 h after pneumococcal challenge. Twenty-four-hour survival (primary outcome), lung injury, bacterial burden, immune and mitochondrial dysfunction, and lung transcriptomes (secondary outcomes) were assessed. RESULTS: High-pressure adverse mechanical ventilation reduced the survival of infected animals (0%; 0 of 7) compared with spontaneous breathing (100%; 7 of 7) and protective mechanical ventilation (86%; 6 of 7; both P < 0.001), with higher lung pathology scores (median [interquartile ranges], 5.5 [4.5 to 7.0] vs. 12.6 [12.0 to 14.0]; P = 0.046), interleukin-8 lung concentrations (106 [54 to 316] vs. 804 [753 to 868] pg/g of lung; P = 0.012), and alveolar mitochondrial DNA release (0.33 [0.28 to 0.36] vs. 0.98 [0.76 to 1.21] ng/µl; P < 0.001) compared with infected spontaneously breathing animals. Survival (0%; 0 of 7; control group) was improved by mesenchymal stem cells (57%; 4 of 7; P = 0.001) or ceftaroline alone (57%; 4 of 7; P < 0.001) and improved even more with a combination treatment (86%; 6 of 7; P < 0.001). Mesenchymal stem cells reduced lung pathology score (8.5 [7.0 to 10.5] vs. 12.6 [12.0 to 14.0]; P = 0.043) and alveolar mitochondrial DNA release (0.39 (0.34 to 0.65) vs. 0.98 (0.76 to 1.21) ng/µl; P = 0.025). Mesenchymal stem cells combined with ceftaroline reduced interleukin-8 lung concentrations (665 [595 to 795] vs. 804 [753 to 868] pg/g of lung; P = 0.007) compared to ceftaroline alone. CONCLUSIONS: In this preclinical study, mesenchymal stem cells improved the outcome of rabbits with pneumonia and high-pressure mechanical ventilation by correcting immune and mitochondrial dysfunction and when combined with the antibiotic ceftaroline was synergistic in mitigating lung inflammation.

Porphyromonas gingivalis Components/Secretions Synergistically Enhance Pneumonia Caused by Streptococcus pneumoniae in Mice.

Streptococcus pneumoniae is an important causative organism of respiratory tract infections. Although periodontal bacteria have been shown to influence respiratory infections such as aspiration pneumonia, the synergistic effect of S. pneumoniae and Porphyromonas gingivalis, a periodontopathic bacterium, on pneumococcal infections is unclear. To investigate whether P. gingivalis accelerates pneumococcal infections, we tested the effects of inoculating P. gingivalis culture supernatant (PgSup) into S. pneumoniae-infected mice. Mice were intratracheally injected with S. pneumoniae and PgSup to induce pneumonia, and lung histopathological sections and the absolute number and frequency of neutrophils and macrophages in the lung were analyzed. Proinflammatory cytokine/chemokine expression was examined by qPCR and ELISA. Inflammatory cell infiltration was observed in S. pneumoniae-infected mice and S. pnemoniae and PgSup mixed-infected mice, and mixed-infected mice showed more pronounced inflammation in lung. The ratios of monocytes/macrophages and neutrophils were not significantly different between the lungs of S. pneumoniae-infected mice and those of mixed-infected mice. PgSup synergistically increased TNF-α expression/production and IL-17 production compared with S. pneumoniae infection alone. We demonstrated that PgSup enhanced inflammation in pneumonia caused by S. pneumoniae, suggesting that virulence factors produced by P. gingivalis are involved in the exacerbation of respiratory tract infections such as aspiration pneumonia.

Host Stress Signals Stimulate Pneumococcal Transition from Colonization to Dissemination into the Lungs.

Streptococcus pneumoniae is an asymptomatic colonizer of the nasopharynx, but it is also one of the most important bacterial pathogens of humans, causing a wide range of mild to life-threatening diseases. The basis of the pneumococcal transition from a commensal to a parasitic lifestyle is not fully understood. We hypothesize that exposure to host catecholamine stress hormones is important for this transition. In this study, we demonstrated that pneumococci preexposed to a hormone released during stress, norepinephrine (NE), have an increased capacity to translocate from the nasopharynx into the lungs compared to untreated pneumococci. Examination of NE-treated pneumococci revealed major alterations in metabolic profiles, cell associations, capsule synthesis, and cell size. By systemically mutating all 12 two-component and 1 orphan regulatory systems, we also identified a unique genetic regulatory circuit involved in pneumococcal recognition and responsiveness to human stress hormones. IMPORTANCE Microbes acquire unique lifestyles under different environmental conditions. Although this is a widespread occurrence, our knowledge of the importance of various host signals and their impact on microbial behavior is not clear despite the therapeutic value of this knowledge. We discovered that catecholamine stress hormones are the host signals that trigger the passage of Streptococcus pneumoniae from a commensal to a parasitic state. We identify that stress hormone treatment of this microbe leads to reductions in cell size and capsule synthesis and renders it more able to migrate from the nasopharynx into the lungs in a mouse model of infection. The microbe requires the TCS09 protein for the recognition and processing of stress hormone signals. Our work has particular clinical significance as catecholamines are abundant in upper respiratory fluids as well as being administered therapeutically to reduce inflammation in ventilated patients, which may explain why intubation in the critically ill is a recognized risk factor for the development of pneumococcal pneumonia.

Inosine monophosphate and inosine differentially regulate endotoxemia and bacterial sepsis.

Inosine monophosphate (IMP) is the intracellular precursor for both adenosine monophosphate and guanosine monophosphate and thus plays a central role in intracellular purine metabolism. IMP can also serve as an extracellular signaling molecule, and can regulate diverse processes such as taste sensation, neutrophil function, and ischemia-reperfusion injury. How IMP regulates inflammation induced by bacterial products or bacteria is unknown. In this study, we demonstrate that IMP suppressed tumor necrosis factor (TNF)-α production and augmented IL-10 production in endotoxemic mice. IMP exerted its effects through metabolism to inosine, as IMP only suppressed TNF-α following its CD73-mediated degradation to inosine in lipopolysaccharide-activated macrophages. Studies with gene targeted mice and pharmacological antagonism indicated that A2A , A2B, and A3 adenosine receptors are not required for the inosine suppression of TNF-α production. The inosine suppression of TNF-α production did not require its metabolism to hypoxanthine through purine nucleoside phosphorylase or its uptake into cells through concentrative nucleoside transporters indicating a role for alternative metabolic/uptake pathways. Inosine augmented IL-ß production by macrophages in which inflammasome was activated by lipopolysaccharide and ATP. In contrast to its effects in endotoxemia, IMP failed to affect the inflammatory response to abdominal sepsis and pneumonia. We conclude that extracellular IMP and inosine differentially regulate the inflammatory response.

Bruton's Tyrosine Kinase-Mediated Signaling in Myeloid Cells Is Required for Protective Innate Immunity During Pneumococcal Pneumonia.

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.

Polyvalent Bacterial Lysate Protects Against Pneumonia Independently of Neutrophils, IL-17A or Caspase-1 Activation.

Polyvalent bacterial lysates have been in use for decades for prevention and treatment of respiratory infections with reported clinical benefits. However, besides claims of broad immune activation, the mode of action is still a matter of debate. The lysates, formulated with the main bacterial species involved in respiratory infections, are commonly prepared by chemical or mechanical disruption of bacterial cells, what is believed influences the biological activity of the product. Here, we prepared two polyvalent lysates with the same composition but different method of bacterial cell disruption and evaluated their biological activity in a comparative fashion. We found that both bacterial lysates induce NF-kB activation in a MyD88 dependent manner, suggesting they work as TLR agonists. Further, we found that a single intranasal dose of any of the two lysates, is sufficient to protect against pneumococcal pneumonia, suggesting that they exert similar biological activity. We have previously shown that protection against pneumococcal pneumonia can also be induced by prior S. pneumoniae sub lethal infection or therapeutic treatment with a TLR5 agonist. Protection in those cases depends on neutrophil recruitment to the lungs, and can be associated with increased local expression of IL-17A. Here, we show that bacterial lysates exert protection against pneumococcal pneumonia independently of neutrophils, IL-17A or Caspase-1/11 activation, suggesting the existence of redundant mechanisms of protection. Trypsin-treated lysates afford protection to the same extent, suggesting that just small peptides suffice to exert the protective effect or that the molecules responsible for the protective effect are not proteins. Understanding the mechanism of action of bacterial lysates and deciphering the active components shall allow redesigning them with more precisely defined formulations and expanding their range of action.

Pulmonary fibrosis in Fra-2 transgenic mice is associated with decreased numbers of alveolar macrophages and increased susceptibility to pneumococcal pneumonia.

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of Streptococcus in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with Streptococcus pneumoniae were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with S. pneumoniae, bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with S. pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1ß, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with S. pneumoniae. Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

Endothelial Cell Protein C Receptor Deficiency Attenuates Streptococcus pneumoniae-induced Pleural Fibrosis.

Streptococcus pneumoniae is the leading cause of hospital community-acquired pneumonia. Patients with pneumococcal pneumonia may develop complicated parapneumonic effusions or empyema that can lead to pleural organization and subsequent fibrosis. The pathogenesis of pleural organization and scarification involves complex interactions between the components of the immune system, coagulation, and fibrinolysis. EPCR (endothelial protein C receptor) is a critical component of the protein C anticoagulant pathway. The present study was performed to evaluate the role of EPCR in the pathogenesis of S. pneumoniae infection-induced pleural thickening and fibrosis. Our studies show that the pleural mesothelium expresses EPCR. Intrapleural instillation of S. pneumoniae impairs lung compliance and lung volume in wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. Intrapleural S. pneumoniae infection induces pleural thickening in wild-type mice. Pleural thickening is more pronounced in EPCR-overexpressing mice, whereas it is reduced in EPCR-deficient mice. Markers of mesomesenchymal transition are increased in the visceral pleura of S. pneumoniae-infected wild-type and EPCR-overexpressing mice but not in EPCR-deficient mice. The lungs of wild-type and EPCR-overexpressing mice administered intrapleural S. pneumoniae showed increased infiltration of macrophages and neutrophils, which was significantly reduced in EPCR-deficient mice. An analysis of bacterial burden in the pleural lavage, the lungs, and blood revealed a significantly lower bacterial burden in EPCR-deficient mice compared with wild-type and EPCR-overexpressing mice. Overall, our data provide strong evidence that EPCR deficiency protects against S. pneumoniae infection-induced impairment of lung function and pleural remodeling.

Proteolytic cleavage of HLA class II by human neutrophil elastase in pneumococcal pneumonia.

Bacterial and viral respiratory infections can initiate acute lung injury and acute respiratory distress syndrome. Neutrophils and their granule enzymes, including neutrophil elastase, are key mediators of the pathophysiology of acute respiratory failure. Although intracellular neutrophil elastase functions as a host defensive factor against pathogens, its leakage into airway spaces induces degradation of host connective tissue components. This leakage disrupts host innate immune responses via proteolytic cleavage of Toll-like receptors and cytokines. Here, we investigated whether neutrophils possess proteases that cleave adaptive immune molecules. We found that expression of the human leukocyte antigen (HLA) class II molecule HLA-DP ß1 was decreased in THP-1-derived macrophages treated with supernatants from dead neutrophils. This decreased HLA-DP ß1 expression was counteracted by treatment with neutrophil elastase inhibitor, suggesting proteolytic cleavage of HLA-DP ß1 by neutrophil elastase. SDS-PAGE showed that neutrophil elastase cleaved recombinant HLA-DP α1, -DP ß1, -DQ α1, -DQ ß1, -DR α, and -DR ß1. Neutrophil elastase also cleaved HLA-DP ß1 on extracellular vesicles isolated from macrophages without triggering morphological changes. Thus, leakage of neutrophil elastase may disrupt innate immune responses, antigen presentation, and T cell activation. Additionally, inhibition of neutrophil elastase is a potential therapeutic option for treating bacterial and viral pneumonia.

Role of the COX2-PGE2 axis in S. pneumoniae-induced exacerbation of experimental fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease (ILD) associated with high morbidity and mortality. Patients with ILD frequently develop an acute exacerbation of their disease, which may be triggered by viral and/or bacterial infections. Prostaglandin E2 (PGE2) is an eicosanoid released in a cyclooxygenase-2 (COX2)-dependent manner and is considered to contribute to regulation of lung fibrosis. However, its role in infection-induced exacerbation of lung fibrosis is poorly defined. We found significantly increased levels of PGE2 in lung tissue of patients with ILD. Increased levels of PGE2 were also found in lung tissue of mice with AdTGF-ß1-induced lung fibrosis and even more so in Streptococcus pneumoniae exacerbated lung fibrosis. Type II alveolar epithelial cells (AT II cells) and alveolar macrophages (AM) contributed to PGE2 release during exacerbating fibrosis. Application of parecoxib to inhibit PGE2 synthesis ameliorated lung fibrosis, whereas intratracheal application of PGE2 worsened lung fibrosis in mice. Both interventions had no effect on S. pneumoniae-exacerbated lung fibrosis. Together, we found that the COX2-PGE2 axis has dual roles in fibrosis that may offset each other: PGE2 helps resolve infection/attenuate inflammation in fibrosis exacerbation but accentuates TGF-ß/AT II cell-mediated fibrosis. These data support the efficacy of COX/PGE2 interventions in the setting of non-exacerbating lung fibrosis.

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA.

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by 1H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27-36 is essential to bind purine- or pyrimidine-based nucleosides. Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.

Estradiol resolves pneumonia via ERß in regulatory T cells.

Current treatments for pneumonia (PNA) are focused on the pathogens. Mortality from PNA-induced acute lung injury (PNA-ALI) remains high, underscoring the need for additional therapeutic targets. Clinical and experimental evidence exists for potential sex differences in PNA survival, with males having higher mortality. In a model of severe pneumococcal PNA, when compared with male mice, age-matched female mice exhibited enhanced resolution characterized by decreased alveolar and lung inflammation and increased numbers of Tregs. Recognizing the critical role of Tregs in lung injury resolution, we evaluated whether improved outcomes in female mice were due to estradiol (E2) effects on Treg biology. E2 promoted a Treg-suppressive phenotype in vitro and resolution of PNA in vivo. Systemic rescue administration of E2 promoted resolution of PNA in male mice independent of lung bacterial clearance. E2 augmented Treg expression of Foxp3, CD25, and GATA3, an effect that required ERß, and not ERα, signaling. Importantly, the in vivo therapeutic effects of E2 were lost in Treg-depleted mice (Foxp3DTR mice). Adoptive transfer of ex vivo E2-treated Tregs rescued Streptococcus pneumoniae-induce PNA-ALI, a salutary effect that required Treg ERß expression. E2/ERß was required for Tregs to control macrophage proinflammatory responses. Our findings support the therapeutic role for E2 in promoting resolution of lung inflammation after PNA via ERß Tregs.

Effect of CARD9 Deficiency on Neutrophil-Mediated Host Defense against Pulmonary Infection with Streptococcus pneumoniae.

Streptococcus pneumoniae is a major causative bacterium of community-acquired pneumonia. Dendritic cell-associated C-type lectin-2 (dectin-2), one of the C-type lectin receptors (CLRs), was previously reported to play a pivotal role in host defense against pneumococcal infection through regulating phagocytosis by neutrophils while not being involved in neutrophil accumulation. In the present study, to elucidate the possible contribution of other CLRs to neutrophil accumulation, we examined the role of caspase recruitment domain-containing protein 9 (CARD9), a common adaptor molecule for signal transduction triggered by CLRs, in neutrophilic inflammatory response against pneumococcal infection. Wild-type (WT), CARD9 knockout (KO), and dectin-2 KO mice were infected intratracheally with pneumococcus, and the infected lungs were histopathologically analyzed to assess neutrophil accumulation at 24 h postinfection. Bronchoalveolar lavage fluids (BALFs) were collected at the same time point to count the neutrophils and assess the production of inflammatory cytokines and chemokines. Neutrophil accumulation was significantly decreased in CARD9 KO mice, but not in dectin-2 KO mice. Tumor necrosis factor alpha (TNF-α), keratinocyte-derived chemokine (KC), and macrophage inflammatory protein-2 (MIP-2) production in BALFs were also attenuated in CARD9 KO mice, but not in dectin-2 KO mice. Production of TNF-α and KC by alveolar macrophages stimulated with pneumococcal culture supernatants was significantly attenuated in CARD9 KO mice, but not in dectin-2 KO mice, compared to that in each group's respective control mice. In addition, pneumococcus-infected CARD9 KO mice showed larger bacterial burdens in the lungs than did WT mice. These data indicate that CARD9 is required for neutrophil migration after pneumococcal infection, as well as inflammatory cytokine and chemokine production by alveolar macrophages, and suggest that a CLR distinct from dectin-2 may be involved in this response.