SARS-CoV-2 ORF8 modulates lung inflammation and clinical disease progression.

The virus severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of the current COVID-19 pandemic. It possesses a large 30 kilobase (kb) genome that encodes structural, non-structural, and accessory proteins. Although not necessary to cause disease, these accessory proteins are known to influence viral replication and pathogenesis. Through the synthesis of novel infectious clones of SARS-CoV-2 that lack one or more of the accessory proteins of the virus, we have found that one of these accessory proteins, ORF8, is critical for the modulation of the host inflammatory response. Mice infected with a SARS-CoV-2 virus lacking ORF8 exhibit increased weight loss and exacerbated macrophage infiltration into the lungs. Additionally, infection of mice with recombinant SARS-CoV-2 viruses encoding ORF8 mutations found in variants of concern reveal that naturally occurring mutations in this protein influence disease severity. Our studies with a virus lacking this ORF8 protein and viruses possessing naturally occurring point mutations in this protein demonstrate that this protein impacts pathogenesis.

Major alteration of Lung Microbiome and the Host Reaction in critically ill COVID-19 Patients with high viral load.

Background: Patients with COVID-19 under invasive mechanical ventilation are at higher risk of developing ventilator-associated pneumonia (VAP), associated with increased healthcare costs, and unfavorable prognosis. The underlying mechanisms of this phenomenon have not been thoroughly dissected. Therefore, this study attempted to bridge this gap by performing a lung microbiota analysis and evaluating the host immune responses that could drive the development of VAP. Materials and methods: In this prospective cohort study, mechanically ventilated patients with confirmed SARS-CoV-2 infection were enrolled. Nasal swabs (NS), endotracheal aspirates (ETA), and blood samples were collected initially within 12 hours of intubation and again at 72 hours post-intubation. Plasma samples underwent cytokine and metabolomic analyses, while NS and ETA samples were sequenced for lung microbiome examination. The cohort was categorized based on the development of VAP. Data analysis was conducted using RStudio version 4.3.1. Results: In a study of 36 COVID-19 patients on mechanical ventilation, significant differences were found in the nasal and pulmonary microbiome, notably in Staphylococcus and Enterobacteriaceae, linked to VAP. Patients with VAP showed a higher SARS-CoV-2 viral load, elevated neutralizing antibodies, and reduced inflammatory cytokines, including IFN-δ, IL-1ß, IL-12p70, IL-18, IL-6, TNF-α, and CCL4. Metabolomic analysis revealed changes in 22 metabolites in non-VAP patients and 27 in VAP patients, highlighting D-Maltose-Lactose, Histidinyl-Glycine, and various phosphatidylcholines, indicating a metabolic predisposition to VAP. Conclusions: This study reveals a critical link between respiratory microbiome alterations and ventilator-associated pneumonia in COVID-19 patients, with elevated SARS-CoV-2 levels and metabolic changes, providing novel insights into the underlying mechanisms of VAP with potential management and prevention implications.

SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

Predictive biomarkers for latent Mycobacterium tuberculosis infection.

Tuberculosis is a leading cause of infectious death worldwide, with almost a fourth of the world's population latently infected with its causative agent, Mycobacterium tuberculosis. Current diagnostic methods are insufficient to differentiate between healthy and latently infected populations. Here, we used a machine learning approach to analyze publicly available proteomic data from saliva and serum in Ethiopia's healthy, latent TB (LTBI) and active TB (ATBI) people. Our analysis discovered a profile of six proteins, Mast Cell Expressed Membrane Protein-1, Hemopexin, Lamin A/C, Small Proline Rich Protein 2F, Immunoglobulin Kappa Variable 4-1, and Voltage Dependent Anion Channel 2 that can precisely differentiate between the healthy and latently infected populations. This data suggests that a combination of six host proteins can serve as accurate biomarkers to diagnose latent infection. This is important for populations living in high-risk areas as it may help in the surveillance and prevention of severe disease.

Major adverse cardiovascular events are associated with necroptosis during severe COVID-19.

BACKGROUND: The mechanisms used by SARS-CoV-2 to induce major adverse cardiac events (MACE) are unknown. Thus, we aimed to determine if SARS-CoV-2 can induce necrotic cell death to promote MACE in patients with severe COVID-19. METHODS: This observational prospective cohort study includes experiments with hamsters and human samples from patients with severe COVID-19. Cytokines and serum biomarkers were analysed in human serum. Cardiac transcriptome analyses were performed in hamsters' hearts. RESULTS: From a cohort of 70 patients, MACE was documented in 26% (18/70). Those who developed MACE had higher Log copies/mL of SARS-CoV-2, troponin-I, and pro-BNP in serum. Also, the elevation of IP-10 and a major decrease in levels of IL-17ɑ, IL-6, and IL-1rɑ were observed. No differences were found in the ability of serum antibodies to neutralise viral spike proteins in pseudoviruses from variants of concern. In hamster models, we found a stark increase in viral titters in the hearts 4 days post-infection. The cardiac transcriptome evaluation resulted in the differential expression of ~ 9% of the total transcripts. Analysis of transcriptional changes in the effectors of necroptosis (mixed lineage kinase domain-like, MLKL) and pyroptosis (gasdermin D) showed necroptosis, but not pyroptosis, to be elevated. An active form of MLKL (phosphorylated MLKL, pMLKL) was elevated in hamster hearts and, most importantly, in the serum of MACE patients. CONCLUSION: SARS-CoV-2 identification in the systemic circulation is associated with MACE and necroptosis activity. The increased pMLKL and Troponin-I indicated the occurrence of necroptosis in the heart and suggested necroptosis effectors could serve as biomarkers and/or therapeutic targets. Trial registration Not applicable.

Necroptosis Drives Major Adverse Cardiovascular Events During Severe COVID-19.

Background The mechanisms used by SARS-CoV-2 to induce major adverse cardiac events (MACE) are unknown. Thus, we aimed to determine if SARS-CoV-2 can infect the heart to kill cardiomyocytes and induce MACE in patients with severe COVID-19. Methods This observational prospective cohort study includes experiments with hamsters and human samples from patients with severe COVID-19. Cytokines and serum biomarkers were analyzed in human serum. Cardiac transcriptome analyses were performed in hamsters' hearts. Results From a cohort of 70 patients, MACE was documented in 26% (18/70). Those who developed MACE had higher Log copies/mL of SARS-CoV-2, troponin-I, and pro-BNP in serum. Also, the elevation of IP-10 and a major decrease in levels of IL-17ɑ, IL-6, and IL-1rɑ were observed. No differences were found in the ability of serum antibodies to neutralize viral spike proteins in pseudoviruses from variants of concern. In hamster models, we found a stark increase in viral titers in the hearts 4 days post-infection. The cardiac transcriptome evaluation resulted in the differential expression of ~ 9% of the total transcripts. Analysis of transcriptional changes of the effectors of necroptosis (mixed lineage kinase domain-like, MLKL) and pyroptosis (gasdermin D) showed necroptosis, but not pyroptosis, to be elevated. Active form of MLKL (phosphorylated MLKL, pMLKL) was elevated in hamster hearts and, most importantly, in the serum of MACE patients. Conclusion SARS-CoV-2 can reach the heart during severe COVID-19 and induce necroptosis in the heart of patients with MACE. Thus, pMLKL could be used as a biomarker of cardiac damage and a therapeutic target. Trial registration: Not applicable.

Gut and lung microbiome profiles in pregnant mice.

In recent years, microbiome research has expanded from the gastrointestinal tract to other host sites previously thought to be abacterial such as the lungs. Yet, the effects of pregnancy in the lung and gut microbiome remains unclear. Here we examined the changes in the gut and lung microbiome in mice at 14 days of gestation. Lung tissue and stool samples were collected from pregnant and non-pregnant female BALB/c mice, DNA was isolated, amplified, and bacterial specific V4 16S rRNA gene was sequenced. Using an in-house bioinformatic pipeline we assessed the microbial composition of each organ using stool and lung tissue samples. The stool data showed that Lachnospiraceae and Lactobacillaceae were more abundant in the pregnant mice. Likewise, Lactobacillaceae were dominant in the lungs of pregnant mice. However, Streptococcaceae were dominant in the lungs of non-pregnant mice with a low microbial abundance in the pregnant mice. A permutation test showed that pregnancy significantly contributes to the variance in both the lung and stool microbiome. At the same time, we estimate that 49% of the total detected operational taxonomic units were shared between the stool and lung data. After removing common stool-associated bacteria from the lung dataset, no microbial differential abundance was detected between the pregnant and non-pregnant lung microbial community. Thus, pregnancy contributes to variance to the lung and stool microbiome but not in the unique lung microbiota.

A multiomics analysis of direct interkingdom dynamics between influenza A virus and Streptococcus pneumoniae uncovers host-independent changes to bacterial virulence fitness.

BACKGROUND: For almost a century, it has been recognized that influenza A virus (IAV) infection can promote the development of secondary bacterial infections (SBI) mainly caused by Streptococcus pneumoniae (Spn). Recent observations have shown that IAV is able to directly bind to the surface of Spn. To gain a foundational understanding of how direct IAV-Spn interaction alters bacterial biological fitness we employed combinatorial multiomic and molecular approaches. RESULTS: Here we show IAV significantly remodels the global transcriptome, proteome and phosphoproteome profiles of Spn independently of host effectors. We identified Spn surface proteins that interact with IAV proteins (hemagglutinin, nucleoprotein, and neuraminidase). In addition, IAV was found to directly modulate expression of Spn virulence determinants such as pneumococcal surface protein A, pneumolysin, and factors associated with antimicrobial resistance among many others. Metabolic pathways were significantly altered leading to changes in Spn growth rate. IAV was also found to drive Spn capsule shedding and the release of pneumococcal surface proteins. Released proteins were found to be involved in evasion of innate immune responses and actively reduced human complement hemolytic and opsonizing activity. IAV also led to phosphorylation changes in Spn proteins associated with metabolism and bacterial virulence. Validation of proteomic data showed significant changes in Spn galactose and glucose metabolism. Furthermore, supplementation with galactose rescued bacterial growth and promoted bacterial invasion, while glucose supplementation led to enhanced pneumolysin production and lung cell apoptosis. CONCLUSIONS: Here we demonstrate that IAV can directly modulate Spn biology without the requirement of host effectors and support the notion that inter-kingdom interactions between human viruses and commensal pathobionts can promote bacterial pathogenesis and microbiome dysbiosis.

An optimized approach for processing of frozen lung and lavage samples for microbiome studies.

The respiratory tract has a resident microbiome with low biomass and limited diversity. This results in difficulties with sample preparation for sequencing due to uneven bacteria-to-host DNA ratio, especially for small tissue samples such as mouse lungs. We compared effectiveness of current procedures used for DNA extraction in microbiome studies. Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected to test different forms of sample pre-treatment and extraction methods to increase bacterial DNA yield and optimize library preparation. DNA extraction using a pre-treatment method of mechanical lysis (lung tissue) and one-step centrifugation (BALF) increased DNA yield and bacterial content of samples. In contrast, a significant increase of environmental contamination was detected after phenol chloroform isoamyl alcohol (PCI) extraction and nested PCR. While PCI has been a standard procedure used in microbiome studies, our data suggests that it is not efficient for DNA extraction of frozen low biomass samples. Finally, a DNA Enrichment kit was tested and found to improve the 16S copy number of lung tissue with a minor shift in microbial composition. Overall, we present a standardized method to provide high yielding DNA and improve sequencing coverage of low microbial biomass frozen samples with minimal contamination.

Pandemic Influenza Infection Promotes Streptococcus pneumoniae Infiltration, Necrotic Damage, and Proteomic Remodeling in the Heart.

For over a century, it has been reported that primary influenza infection promotes the development of a lethal form of bacterial pulmonary disease. More recently, pneumonia events caused by both viruses and bacteria have been directly associated with cardiac damage. Importantly, it is not known whether viral-bacterial synergy extends to extrapulmonary organs such as the heart. Using label-free quantitative proteomics and molecular approaches, we report that primary infection with pandemic influenza A virus leads to increased Streptococcus pneumoniae translocation to the myocardium, leading to general biological alterations. We also observed that each infection alone led to proteomic changes in the heart, and these were exacerbated in the secondary bacterial infection (SBI) model. Gene ontology analysis of significantly upregulated proteins showed increased innate immune activity, oxidative processes, and changes to ion homeostasis during SBI. Immunoblots confirmed increased complement and antioxidant activity in addition to increased expression of angiotensin-converting enzyme 2. Using an in vitro model of sequential infection in human cardiomyocytes, we observed that influenza enhances S. pneumoniae cytotoxicity by promoting oxidative stress enhancing bacterial toxin-induced necrotic cell death. Influenza infection was found to increase receptors that promote bacterial adhesion, such as polymeric immunoglobulin receptor and fibronectin leucine-rich transmembrane protein 1 in cardiomyocytes. Finally, mice deficient in programmed necrosis (i.e., necroptosis) showed enhanced innate immune responses, decreased virus-associated pathways, and promotion of mitochondrial function upon SBI. The presented results provide the first in vivo evidence that influenza infection promotes S. pneumoniae infiltration, necrotic damage, and proteomic remodeling of the heart. IMPORTANCE Adverse cardiac events are a common complication of viral and bacterial pneumonia. For over a century, it has been recognized that influenza infection promotes severe forms of pulmonary disease mainly caused by the bacterium Streptococcus pneumoniae. The extrapulmonary effects of secondary bacterial infections to influenza virus are not known. In the present study, we used a combination of quantitative proteomics and molecular approaches to assess the underlying mechanisms of how influenza infection promotes bacteria-driven cardiac damage and proteome remodeling. We further observed that programmed necrosis (i.e., necroptosis) inhibition leads to reduced damage and proteome changes associated with health.

Kinetic Multi-omic Analysis of Responses to SARS-CoV-2 Infection in a Model of Severe COVID-19.

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is poorly understood due to a lack of an animal model that recapitulates severe human disease. Here, we report a Syrian hamster model that develops progressive lethal pulmonary disease that closely mimics severe coronavirus disease 2019 (COVID-19). We evaluated host responses using a multi-omic, multiorgan approach to define proteome, phosphoproteome, and transcriptome changes. These data revealed both type I and type II interferon-stimulated gene and protein expression along with a progressive increase in chemokines, monocytes, and neutrophil-associated molecules throughout the course of infection that peaked in the later time points correlating with a rapidly developing diffuse alveolar destruction and pneumonia that persisted in the absence of active viral infection. Extrapulmonary proteome and phosphoproteome remodeling was detected in the heart and kidneys following viral infection. Together, our results provide a kinetic overview of multiorgan host responses to severe SARS-CoV-2 infection in vivo. IMPORTANCE The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has created an urgent need to understand the pathogenesis of this infection. These efforts have been impaired by the lack of animal models that recapitulate severe coronavirus disease 2019 (COVID-19). Here, we report a hamster model that develops severe COVID-19-like disease following infection with human isolates of SARS-CoV-2. To better understand pathogenesis, we evaluated changes in gene transcription and protein expression over the course of infection to provide an integrated multiorgan kinetic analysis of the host response to infection. These data reveal a dynamic innate immune response to infection and corresponding immune pathologies consistent with severe human disease. Altogether, this model will be useful for understanding the pathogenesis of severe COVID-19 and for testing interventions.

Streptococcus pneumoniae binds to host GAPDH on dying lung epithelial cells worsening secondary infection following influenza.

Streptococcus pneumoniae (Spn) alone and during co-infection with influenza A virus (IAV) can result in severe pneumonia with mortality. Pneumococcal surface protein A (PspA) is an established virulence factor required for Spn evasion of lactoferricin and C-reactive protein-activated complement-mediated killing. Herein, we show that PspA functions as an adhesin to dying host cells. We demonstrate that PspA binds to host-derived glyceraldehyde-3-phosphate dehydrogenase (GAPDH) bound to outward-flipped phosphatidylserine residues on dying host cells. PspA-mediated adhesion was to apoptotic, pyroptotic, and necroptotic cells, but not healthy lung cells. Using isogenic mutants of Spn, we show that PspA-GAPDH-mediated binding to lung cells increases pneumococcal localization in the lower airway, and this is enhanced as a result of pneumolysin exposure or co-infection with IAV. PspA-mediated binding to GAPDH requires amino acids 230-281 in its α-helical domain with intratracheal inoculation of this PspA fragment alongside the bacteria reducing disease severity in an IAV/Spn pneumonia model.

Streptococcus pneumoniae Binds to Host Lactate Dehydrogenase via PspA and PspC To Enhance Virulence.

Pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC, also called CbpA) are major virulence factors of Streptococcus pneumoniae (Spn). These surface-exposed choline-binding proteins (CBPs) function independently to inhibit opsonization, neutralize antimicrobial factors, or serve as adhesins. PspA and PspC both carry a proline-rich domain (PRD) whose role, other than serving as a flexible connector between the N-terminal and C-terminal domains, was up to this point unknown. Herein, we demonstrate that PspA binds to lactate dehydrogenase (LDH) released from dying host cells during infection. Using recombinant versions of PspA and isogenic mutants lacking PspA or specific domains of PspA, this property was mapped to a conserved 22-amino-acid nonproline block (NPB) found within the PRD of most PspAs and PspCs. The NPB of PspA had specific affinity for LDH-A, which converts pyruvate to lactate. In a mouse model of pneumonia, preincubation of Spn carrying NPB-bearing PspA with LDH-A resulted in increased bacterial titers in the lungs. In contrast, incubation of Spn carrying a version of PspA lacking the NPB with LDH-A or incubation of wild-type Spn with enzymatically inactive LDH-A did not enhance virulence. Preincubation of NPB-bearing Spn with lactate alone enhanced virulence in a pneumonia model, indicating exogenous lactate production by Spn-bound LDH-A had an important role in pneumococcal pathogenesis. Our observations show that lung LDH, released during the infection, is an important binding target for Spn via PspA/PspC and that pneumococci utilize LDH-A derived lactate for their benefit in vivoIMPORTANCEStreptococcus pneumoniae (Spn) is the leading cause of community-acquired pneumonia. PspA and PspC are among its most important virulence factors, and these surface proteins carry the proline-rich domain (PRD), whose role was unknown until now. Herein, we show that a conserved 22-amino-acid nonproline block (NPB) found within most versions of the PRD binds to host-derived lactate dehydrogenase A (LDH-A), a metabolic enzyme which converts pyruvate to lactate. PspA-mediated binding of LDH-A increased Spn titers in the lungs and this required LDH-A enzymatic activity. Enhanced virulence was also observed when Spn was preincubated with lactate, suggesting LDH-A-derived lactate is a vital food source. Our findings define a role for the NPB of the PRD and show that Spn co-opts host enzymes for its benefit. They advance our understanding of pneumococcal pathogenesis and have key implications on the susceptibility of individuals with preexisting airway damage that results in LDH-A release.

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.

Global Proteome and Phosphoproteome Characterization of Sepsis-induced Kidney Injury.

Sepsis-induced acute kidney injury (S-AKI) is the most common complication in hospitalized and critically ill patients, highlighted by a rapid decline of kidney function occurring a few hours or days after sepsis onset. Systemic inflammation elicited by microbial infections is believed to lead to kidney damage under immunocompromised conditions. However, although AKI has been recognized as a disease with long-term sequelae, partly because of the associated higher risk of chronic kidney disease (CKD), the understanding of kidney pathophysiology at the molecular level and the global view of dynamic regulations in situ after S-AKI, including the transition to CKD, remains limited. Existing studies of S-AKI mainly focus on deriving sepsis biomarkers from body fluids. In the present study, we constructed a mid-severity septic murine model using cecal ligation and puncture (CLP), and examined the temporal changes to the kidney proteome and phosphoproteome at day 2 and day 7 after CLP surgery, corresponding to S-AKI and the transition to CKD, respectively, by employing an ultrafast and economical filter-based sample processing method combined with the label-free quantitation approach. Collectively, we identified 2,119 proteins and 2950 phosphosites through multi-proteomics analyses. Among them, we identified an array of highly promising candidate marker proteins indicative of disease onset and progression accompanied by immunoblot validations, and further denoted the pathways that are specifically responsive to S-AKI and its transition to CKD, which include regulation of cell metabolism regulation, oxidative stress, and energy consumption in the diseased kidneys. Our data can serve as an enriched resource for the identification of mechanisms and biomarkers for sepsis-induced kidney diseases.

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.

Omega-3 fatty acids in contrast to omega-6 protect against pneumococcal pneumonia.

PURPOSE: The aim of this study was to assess if long-term supplementation of omega-3 fatty acids or a diet rich in omega-6 fatty acids ameliorates disease severity in a murine model of pneumococcal pneumonia. We hypothesize that long-term dietary supplementation of omega-3 fatty acids will reduce inflammation, disease severity and improve survival compared to omega-6 fatty acids. METHODS: Mice receiving diets supplemented with Omega-3 or Omega-6 for two months were intranasally infected with Streptococcus pneumoniae. We analyzed survival, bacterial burden, histopathology and inflammatory biomarkers. RESULTS: Our results showed that Omega-3 supplementation had increased survival (p = 0.005), less bacteremia (p = 0.0001) and lower bacterial burden in the lungs (p = 0.0002) when compared to the Omega-6 supplementation. Overall, Omega-3 reduced lung pathology, in particular peribronchial inflammation and cell death. Analyses of lung homogenates showed the Omega-3 cohort had decreased levels of the inflammatory cytokine interleukin-6 and an increase in anti-inflammatory cytokine interleukin-10. CONCLUSIONS: Supplementation of mouse diets with Omega-3 fatty acids improved survival, bacterial invasion in the blood and lungs as well as decreased overall lung tissue inflammation and cell death when compared to the Omega-6 supplemented diets. Translation of these findings in humans may improve outcomes of patients at risk for pneumonia.

NAD hydrolysis by the tuberculosis necrotizing toxin induces lethal oxidative stress in macrophages.

Mycobacterium tuberculosis (Mtb) kills infected macrophages through necroptosis, a programmed cell death that enhances mycobacterial replication and dissemination. The tuberculosis necrotizing toxin (TNT) is the major cytotoxicity factor of Mtb in macrophages and induces necroptosis by NAD+ hydrolysis. Here, we show that the catalytic activity of TNT triggers the production of reactive oxygen species (ROS) in Mtb-infected macrophages causing cell death and promoting mycobacterial replication. TNT induces ROS formation both by activating necroptosis and by a necroptosis-independent mechanism. Most of the detected ROS originate in mitochondria as a consequence of opening the mitochondrial permeability transition pore. However, a significant part of ROS is produced by mechanisms independent of TNT and necroptosis. Expressing only the tnt gene in Jurkat T-cells also induces lethal ROS formation indicating that these molecular mechanisms are not restricted to macrophages. Both the antioxidant N-acetyl-cysteine and replenishment of NAD+ by providing nicotinamide reduce ROS levels in Mtb-infected macrophages, protect them from cell death, and restrict mycobacterial replication. Our results indicate that a host-directed therapy combining replenishment of NAD+ with inhibition of necroptosis and/or antioxidants might improve the health status of TB patients and augment antibacterial TB chemotherapy.