Influenza Causes MLKL-Driven Cardiac Proteome Remodeling During Convalescence.

RATIONALE: Patients with and without cardiovascular diseases have been shown to be at risk of influenza-mediated cardiac complications. Recent clinical reports support the notion of a direct link between laboratory-confirmed influenza virus infections and adverse cardiac events. OBJECTIVE: Define the molecular mechanisms underlying influenza virus-induced cardiac pathogenesis after resolution of pulmonary infection and the role of necroptosis in this process. METHODS AND RESULTS: Hearts from wild-type and necroptosis-deficient (MLKL [mixed lineage kinase domain-like protein]-KO) mice were dissected 12 days after initial influenza A virus (IAV) infection when viral titers were undetectable in the lungs. Immunofluorescence microscopy and plaque assays showed presence of viable IAV particles in the myocardium without generation of interferon responses. Global proteome and phosphoproteome analyses using high-resolution accurate mass-based LC-MS/MS and label-free quantitation showed that the global proteome as well as the phosphoproteome profiles were significantly altered in IAV-infected mouse hearts in a strain-independent manner. Necroptosis-deficient mice had increased survival and reduced weight loss post-IAV infection, as well as increased antioxidant and mitochondrial function, indicating partial protection to IAV infection. These findings were confirmed in vitro by pretreatment of human and rat myocytes with antioxidants or necroptosis inhibitors, which blunted oxidative stress and mitochondrial damage after IAV infection. CONCLUSIONS: This study provides the first evidence that the cardiac proteome and phosphoproteome are significantly altered post-pulmonary influenza infection. Moreover, viral particles can persist in the heart after lung clearance, altering mitochondrial function and promoting cell death without active replication and interferon responses. Finally, our findings show inhibition of necroptosis or prevention of mitochondrial damage as possible therapeutic interventions to reduce cardiac damage during influenza infections. Graphic Abstract: A graphic abstract is available for this article.

Influenza-Induced Oxidative Stress Sensitizes Lung Cells to Bacterial-Toxin-Mediated Necroptosis.

Pneumonias caused by influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity and high mortality rate. Previously, we have shown that bacterial pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondary Streptococcus pneumoniae (Spn) infection. We observe that IAV synergistically sensitizes lung epithelial cells for PFT-mediated necroptosis in vitro and in murine models of Spn co-infection and secondary infection. Pharmacoelogical induction of oxidative stress without virus sensitizes cells for PFT-mediated necroptosis. Antioxidant treatment or inhibition of necroptosis reduces disease severity during secondary bacterial infection. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identify necroptosis inhibition and antioxidant therapy as potential intervention strategies.

Inhibition of Necroptosis to Prevent Long-term Cardiac Damage During Pneumococcal Pneumonia and Invasive Disease.

BACKGROUND: Streptococcus pneumoniae infection can result in bacteremia with devastating consequences including heart damage. Necroptosis is a proinflammatory form of cell death instigated by pore-forming toxins such as S. pneumoniae pneumolysin. Necroptosis-inhibiting drugs may lessen organ damage during invasive pneumococcal disease (IPD). METHODS: In vitro experiments were carried out with human and mouse cardiomyocytes. Long-term cardiac damage was assessed using high-resolution echocardiography in ampicillin-rescued mice 3 months after challenge with S. pneumoniae. Ponatinib, a necroptosis-inhibiting and Food and Drug Administration-approved drug for lymphocytic leukemia treatment, was administered intraperitoneally alongside ampicillin to test its therapeutic efficacy. Histology of heart sections included hematoxylin-eosin staining for overt damage, immunofluorescence for necroptosis, and Sirius red/fast green staining for collagen deposition. RESULTS: Cardiomyocyte death and heart damage was due to pneumolysin-mediated necroptosis. IPD leads to long-term cardiac damage, as evidenced by de novo collagen deposition in mouse hearts and a decrease in fractional shortening. Adjunct necroptosis inhibition reduced the number of S. pneumoniae foci observed in hearts of acutely infected mice and serum levels of troponin I. Ponatinib reduced collagen deposition and protected heart function in convalescence. CONCLUSIONS: Acute and long-term cardiac damage incurred during IPD is due in part to cardiomyocyte necroptosis. Necroptosis inhibitors may be a viable adjunct therapy.

Checkpoint blockade inhibitors enhances the effectiveness of a Listeria monocytogenes-based melanoma vaccine.

Melanoma continues to be a significant health concern worldwide despite recent improvements in treatment. Unlike many other prominent cancers, melanoma incidence in both men and women increased over the past decade in the U. S. and much of the developed world. The single greatest risk factor for melanoma is damage from ultraviolet radiation associated with lifestyle. The lifestyle component suggests that although melanoma risk can be minimized with behavioral changes, vaccinating high-risk individuals against melanoma may be the most efficacious preventative method. Accordingly, using a highly attenuated, double-mutant L. monocytogenes strain expressing a tumor-associated antigen, we obtained significant protection against melanoma in a mouse model. The Listeria-based vaccine induced protection through antigen-specific CD8+ T-cells inducing both a protective primary and a memory T-cell response. Vaccinated animals were significantly protected from melanoma. When used in conjunction with checkpoint blockade treatment, the vaccine substantially reduced tumor size and number relative to animals receiving checkpoint blockade (CPB) alone. This study provides evidence that CPB treatment synergizes with a L. monocytogenes-based melanoma vaccine to enhance vaccine-mediated protection.

House dust mite exposure attenuates influenza A infection in a mouse model of pulmonary allergic inflammation.

Environmental allergens elicit complex immune responses in the lungs that can promote the development of asthma or exacerbate preexisting asthma in susceptible individuals. House dust mites are one of the most common indoor allergens and are a significant driver of allergic disease. Respiratory infections are known factors in acute exacerbations of asthma but the impact of allergen on the pathogen is not well understood. We investigated the pathogenesis of influenza A infection following exposure to house dust mites. Mice exposed to house dust mites lose less weight following infection and had more transcription of interferon-lambda than controls. These data correlated with less transcription of the influenza polymerase acidic gene suggesting diminished viral replication in house dust mite exposed mice. Altogether, these data suggest that exposure to environmental allergens can influence the pathogenesis of influenza infection.

Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1.

Necroptosis complements apoptosis as a host defense pathway to stop virus infection. Herpes simplex virus shows a propensity to trigger necroptosis of mouse cells and mice even though cell death is blocked in human cells through UL39-encoded ICP6. This ribonucleotide reductase large subunit (R1) nucleates RHIM-dependent oligomerization of RIP3 kinase (RIPK3, also known as RIP3) in mouse cells but inhibits activation in cells from the natural human host. By interrogating the comparative behavior of ICP6-deficient viruses in mouse and human cells, here we unveil virus-induced necroptosis mediated by Z-DNA-binding protein 1 (ZBP1, also known as DAI). ZBP1 acts as a pathogen sensor to detect nascent RNA transcripts rather than input viral DNA or viral DNA generated through replication. Consistent with the implicated role of virus-induced necroptosis in restricting infection, viral pathogenesis is restored in Zbp1-/-, Ripk3-/- and Mlkl-/- mice. Thus, in addition to direct activation of RIPK3 via ICP6, HSV1 infection in mice and mouse cells triggers virus-induced necroptosis through ZBP1. Importantly, virus-induced necroptosis is also induced in human HT-29 cells by ICP6 mutant viruses; however, ZBP1 levels must be elevated for this pathway to be active. Thus, our studies reveal a common, species-independent role of this nucleic acid sensor to detect the presence of this virus. HSV1 ICP6 functions as a bona fide RHIM signaling inhibitor to block virus-induced necroptosis in its natural host. Altogether, ZBP1-dependent restriction of herpesvirus infection emerges as a potent antiviral armament of the innate immune system.

Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing.

For over 130 years, invasive pneumococcal disease has been associated with the presence of extracellular planktonic pneumococci, i.e. diplococci or short chains in affected tissues. Herein, we show that Streptococcus pneumoniae that invade the myocardium instead replicate within cellular vesicles and transition into non-purulent biofilms. Pneumococci within mature cardiac microlesions exhibited salient biofilm features including intrinsic resistance to antibiotic killing and the presence of an extracellular matrix. Dual RNA-seq and subsequent principal component analyses of heart- and blood-isolated pneumococci confirmed the biofilm phenotype in vivo and revealed stark anatomical site-specific differences in virulence gene expression; the latter having major implications on future vaccine antigen selection. Our RNA-seq approach also identified three genomic islands as exclusively expressed in vivo. Deletion of one such island, Region of Diversity 12, resulted in a biofilm-deficient and highly inflammogenic phenotype within the heart; indicating a possible link between the biofilm phenotype and a dampened host-response. We subsequently determined that biofilm pneumococci released greater amounts of the toxin pneumolysin than did planktonic or RD12 deficient pneumococci. This allowed heart-invaded wildtype pneumococci to kill resident cardiac macrophages and subsequently subvert cytokine/chemokine production and neutrophil infiltration into the myocardium. This is the first report for pneumococcal biofilm formation in an invasive disease setting. We show that biofilm pneumococci actively suppress the host response through pneumolysin-mediated immune cell killing. As such, our findings contradict the emerging notion that biofilm pneumococci are passively immunoquiescent.

Murine cytomegalovirus IE3-dependent transcription is required for DAI/ZBP1-mediated necroptosis.

DNA-dependent activator of interferon regulatory factors/Z-DNA binding protein 1 (DAI/ZBP1) is a crucial sensor of necroptotic cell death induced by murine cytomegalovirus (MCMV) in its natural host. Here, we show that viral capsid transport to the nucleus and subsequent viral IE3-dependent early transcription are required for necroptosis. Necroptosis induction does not depend on input virion DNA or newly synthesized viral DNA A putative RNA-binding domain of DAI/ZBP1, Zα2, is required to sense virus and trigger necroptosis. Thus, MCMV IE3-dependent transcription from the viral genome plays a crucial role in activating DAI/ZBP1-dependent necroptosis. This implicates RNA transcripts generated by a large double-stranded DNA virus as a biologically relevant ligand for DAI/ZBP1 during natural viral infection.

A RIPtide Protects Neurons from Infection.

RIPK3 and RIPK1 limit virus spread by executing either apoptotic or necroptotic cell death in response to infection. In a recent issue of Cell, Daniels et al. (2017) unveil an unexpected cell death-independent requirement of RIP kinase activity in coordinating neuroinflammation, restricting West Nile virus pathogenesis in neurons.

Transcriptional organization of pneumococcal psrP-secY2A2 and impact of GtfA and GtfB deletion on PsrP-associated virulence properties.

Pneumococcal serine-rich repeat protein (PsrP) is a glycoprotein that mediates Streptococcus pneumoniae attachment to lung cells and promotes biofilm formation. Herein, we investigated the transcriptional organization of psrP-secY2A2, the 37-kbp pathogenicity island encoding PsrP and its accessory genes. PCR amplification of cDNA and RNA-seq analysis found psrP-secY2A2 to be minimally composed of three operons: psrP-glyA, glyB, and glyC-asp5. Transcription of all three operons was greatest during biofilm growth and immunoblot analyses confirmed increased PsrP production by biofilm pneumococci. Using gas chromatography-mass spectrometry we identified monomeric N-acetylglucosamine as the primary glycoconjugate present on a recombinant intracellular version of PsrP, i.e. PsrP1-734. This finding was validated by immunoblot using lectins with known carbohydrate specificities. We subsequently deleted gtfA and gtfB, the GTFs thought to be responsible for addition of O-linked N-acetylglucosamine, and tested for PsrP and its associated virulence properties. These deletions negatively affected our ability to detect PsrP1-734 in bacterial whole cell lysates. Moreover, S. pneumoniae mutants lacking these genes pheno-copied the psrP mutant and were attenuated for: biofilm formation, adhesion to lung epithelial cells, and pneumonia in mice. Our studies identify the transcriptional organization of psrP-secY2A2 and show the indispensable role of GtfA and GtfB on PsrP-mediated pneumococcal virulence.

A Non-Human Primate Model of Severe Pneumococcal Pneumonia.

RATIONALE: Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and infectious death in adults worldwide. A non-human primate model is needed to study the molecular mechanisms that underlie the development of severe pneumonia, identify diagnostic tools, explore potential therapeutic targets, and test clinical interventions during pneumococcal pneumonia. OBJECTIVE: To develop a non-human primate model of pneumococcal pneumonia. METHODS: Seven adult baboons (Papio cynocephalus) were surgically tethered to a continuous monitoring system that recorded heart rate, temperature, and electrocardiography. Animals were inoculated with 109 colony-forming units of S. pneumoniae using bronchoscopy. Three baboons were rescued with intravenous ampicillin therapy. Pneumonia was diagnosed using lung ultrasonography and ex vivo confirmation by histopathology and immunodetection of pneumococcal capsule. Organ failure, using serum biomarkers and quantification of bacteremia, was assessed daily. RESULTS: Challenged animals developed signs and symptoms of pneumonia 4 days after infection. Infection was characterized by the presence of cough, tachypnea, dyspnea, tachycardia and fever. All animals developed leukocytosis and bacteremia 24 hours after infection. A severe inflammatory reaction was detected by elevation of serum cytokines, including Interleukin (IL)1Ra, IL-6, and IL-8, after infection. Lung ultrasonography precisely detected the lobes with pneumonia that were later confirmed by pathological analysis. Lung pathology positively correlated with disease severity. Antimicrobial therapy rapidly reversed symptomology and reduced serum cytokines. CONCLUSIONS: We have developed a novel animal model for severe pneumococcal pneumonia that mimics the clinical presentation, inflammatory response, and infection kinetics seen in humans. This is a novel model to test vaccines and treatments, measure biomarkers to diagnose pneumonia, and predict outcomes.

Endothelial adhesion molecules and multiple organ failure in patients with severe sepsis.

OBJECTIVE: To determine if serum levels of endothelial adhesion molecules were associated with the development of multiple organ failure (MOF) and in-hospital mortality in adult patients with severe sepsis. DESIGN: This study was a secondary data analysis of a prospective cohort study. SETTING: Patients were admitted to two tertiary intensive care units in San Antonio, TX, between 2007 and 2012. PATIENTS: Patients with severe sepsis at the time of intensive care unit (ICU) admission were enrolled. Inclusion criteria were consistent with previously published criteria for severe sepsis or septic shock in adults. Exclusion criteria included immunosuppressive medications or conditions. INTERVENTIONS: None. MEASUREMENTS: Baseline serum levels of the following endothelial cell adhesion molecules were measured within the first 72h of ICU admission: Intracellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1), and Vascular Endothelial Growth Factor (VEGF). The primary and secondary outcomes were development of MOF (⩾2 organ dysfunction) and in-hospital mortality, respectively. MAIN RESULTS: Forty-eight patients were enrolled in this study, of which 29 (60%) developed MOF. Patients that developed MOF had higher levels of VCAM-1 (p=0.01) and ICAM-1 (p=0.01), but not VEGF (p=0.70) compared with patients without MOF (single organ failure only). The area under the curve (AUC) to predict MOF according to VCAM-1, ICAM-1 and VEGF was 0.71, 0.73, and 0.54, respectively. Only increased VCAM-1 levels were associated with in-hospital mortality (p=0.03). These associations were maintained even after adjusting for APACHE and SOFA scores using logistic regression. CONCLUSIONS: High levels of serum ICAM-1 was associated with the development of MOF. High levels of VCAM-1 was associated with both MOF and in-hospital mortality.

Neuraminidase A-Exposed Galactose Promotes Streptococcus pneumoniae Biofilm Formation during Colonization.

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and ß-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role.

Infiltrated Macrophages Die of Pneumolysin-Mediated Necroptosis following Pneumococcal Myocardial Invasion.

Streptococcus pneumoniae (the pneumococcus) is capable of invading the heart. Herein we observed that pneumococcal invasion of the myocardium occurred soon after development of bacteremia and was continuous thereafter. Using immunofluorescence microscopy (IFM), we observed that S. pneumoniae replication within the heart preceded visual signs of tissue damage in cardiac tissue sections stained with hematoxylin and eosin. Different S. pneumoniae strains caused distinct cardiac pathologies: strain TIGR4, a serotype 4 isolate, caused discrete pneumococcus-filled microscopic lesions (microlesions), whereas strain D39, a serotype 2 isolate, was, in most instances, detectable only using IFM and was associated with foci of cardiomyocyte hydropic degeneration and immune cell infiltration. Both strains efficiently invaded the myocardium, but cardiac damage was entirely dependent on the pore-forming toxin pneumolysin only for D39. Early microlesions caused by TIGR4 and microlesions formed by a TIGR4 pneumolysin-deficient mutant were infiltrated with CD11b(+) and Ly6G-positive neutrophils and CD11b(+) and F4/80-positive (F4/80(+)) macrophages. We subsequently demonstrated that macrophages in TIGR4-infected hearts died as a result of pneumolysin-induced necroptosis. The effector of necroptosis, phosphorylated mixed-lineage kinase domain-like protein (MLKL), was detected in CD11b(+) and F4/80(+) cells associated with microlesions. Likewise, treatment of infected mice and THP-1 macrophages in vitro with the receptor-interacting protein 1 kinase (RIP1) inhibitor necrostatin-5 promoted the formation of purulent microlesions and blocked cell death, respectively. We conclude that pneumococci that have invaded the myocardium are an important cause of cardiac damage, pneumolysin contributes to cardiac damage in a bacterial strain-specific manner, and pneumolysin kills infiltrated macrophages via necroptosis, which alters the immune response.

Pneumococci in biofilms are non-invasive: implications on nasopharyngeal colonization.

Streptococcus pneumoniae (the pneumococcus) is an opportunistic pathogen that colonizes the human nasopharynx asymptomatically. Invasive pneumococcal disease develops following bacterial aspiration into the lungs. Pneumococci within the nasopharynx exist as biofilms, a growth phenotype characterized by surface attachment, encasement within an extracellular matrix, and antimicrobial resistance. Experimental evidence indicates that biofilm pneumococci are attenuated vs. their planktonic counterpart. Biofilm pneumococci failed to cause invasive disease in experimentally challenged mice and in vitro were shown to be non-invasive despite being hyper-adhesive. This attenuated phenotype corresponds with observations that biofilm pneumococci elicit significantly less cytokine and chemokine production from host cells than their planktonic counterparts. Microarray and proteomic studies show that pneumococci within biofilms have decreased metabolism, less capsular polysaccharide, and reduced production of the pore-forming toxin pneumolysin. Biofilm pneumococci are predominately in the transparent phenotype, which has elevated cell wall phosphorylcholine, an adhesin subject to C-reactive protein mediated opsonization. Herein, we review these changes in virulence, interpret their impact on colonization and transmission, and discuss the notion that non-invasive biofilms are principal lifestyle of S. pneumoniae.

Lipidation of the FPI protein IglE contributes to Francisella tularensis ssp. novicida intramacrophage replication and virulence.

Francisella tularensis is a Gram-negative bacterium responsible for the human disease tularemia. The Francisella pathogenicity island (FPI) encodes a secretion system related to type VI secretion systems (T6SS) which allows F. tularensis to escape the phagosome and replicate within the cytosol of infected macrophages and ultimately cause disease. A lipoprotein is typically found encoded within T6SS gene clusters and is believed to anchor portions of the secretion apparatus to the outer membrane. We show that the FPI protein IglE is a lipoprotein that incorporates (3)H-palmitate and localizes to the outer membrane. A C22G IglE mutant failed to be lipidated and failed to localize to the outer membrane, consistent with C22 being the site of lipidation. Francisella tularensis ssp. novicida expressing IglE C22G is defective for replication in macrophages and unable to cause disease in mice. Bacterial two-hybrid analysis demonstrated that IglE interacts with the C-terminal portion of the FPI inner membrane protein PdpB, and PhoA fusion analysis indicated the PdpB C-terminus is located within the periplasm. We predict this interaction facilitates channel formation to allow secretion through this system.