Amyloid-ß and caspase-1 are indicators of sepsis and organ injury.

Background: Sepsis is a life-threatening condition that results from a dysregulated host response to infection, leading to organ dysfunction. Despite the prevalence and associated socioeconomic costs, treatment of sepsis remains limited to antibiotics and supportive care, and a majority of intensive care unit (ICU) survivors develop long-term cognitive complications post-discharge. The present study identifies a novel regulatory relationship between amyloid-ß (Aß) and the inflammasome-caspase-1 axis as key innate immune mediators that define sepsis outcomes. Methods: Medical ICU patients and healthy individuals were consented for blood and clinical data collection. Plasma cytokine, caspase-1 and Aß levels were measured. Data were compared against indices of multiorgan injury and other clinical parameters. Additionally, recombinant proteins were tested in vitro to examine the effect of caspase-1 on a functional hallmark of Aß, namely aggregation. Results: Plasma caspase-1 levels displayed the best predictive value in discriminating ICU patients with sepsis from non-infected ICU patients (area under the receiver operating characteristic curve=0.7080). Plasma caspase-1 and the Aß isoform Aßx-40 showed a significant positive correlation and Aßx-40 associated with organ injury. Additionally, Aß plasma levels continued to rise from time of ICU admission to 7 days post-admission. In silico, Aß harbours a predicted caspase-1 cleavage site, and in vitro studies demonstrated that caspase-1 cleaved Aß to inhibit its auto-aggregation, suggesting a novel regulatory relationship. Conclusions: Aßx-40 and caspase-1 are potentially useful early indicators of sepsis and its attendant organ injury. Additionally, Aßx-40 has emerged as a potential culprit in the ensuing development of post-ICU syndrome.

Gamma secretase activating protein promotes end-organ dysfunction after bacterial pneumonia.

Pneumonia elicits the production of cytotoxic beta amyloid (Aß) that contributes to end-organ dysfunction, yet the mechanism(s) linking infection to activation of the amyloidogenic pathway that produces cytotoxic Aß is unknown. Here, we tested the hypothesis that gamma-secretase activating protein (GSAP), which contributes to the amyloidogenic pathway in the brain, promotes end-organ dysfunction following bacterial pneumonia. First-in-kind Gsap knockout rats were generated. Wild-type and knockout rats possessed similar body weights, organ weights, circulating blood cell counts, arterial blood gases, and cardiac indices at baseline. Intratracheal Pseudomonas aeruginosa infection caused acute lung injury and a hyperdynamic circulatory state. Whereas infection led to arterial hypoxemia in wild-type rats, the alveolar-capillary barrier integrity was preserved in Gsap knockout rats. Infection potentiated myocardial infarction following ischemia-reperfusion injury, and this potentiation was abolished in knockout rats. In the hippocampus, GSAP contributed to both pre- and postsynaptic neurotransmission, increasing the presynaptic action potential recruitment, decreasing neurotransmitter release probability, decreasing the postsynaptic response, and preventing postsynaptic hyperexcitability, resulting in greater early long-term potentiation but reduced late long-term potentiation. Infection abolished early and late long-term potentiation in wild-type rats, whereas the late long-term potentiation was partially preserved in Gsap knockout rats. Furthermore, hippocampi from knockout rats, and both the wild-type and knockout rats following infection, exhibited a GSAP-dependent increase in neurotransmitter release probability and postsynaptic hyperexcitability. These results elucidate an unappreciated role for GSAP in innate immunity and highlight the contribution of GSAP to end-organ dysfunction during infection.NEW & NOTEWORTHY Pneumonia is a common cause of end-organ dysfunction, both during and in the aftermath of infection. In particular, pneumonia is a common cause of lung injury, increased risk of myocardial infarction, and neurocognitive dysfunction, although the mechanisms responsible for such increased risk are unknown. Here, we reveal that gamma-secretase activating protein, which contributes to the amyloidogenic pathway, is important for end-organ dysfunction following infection.

Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells).

To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR).

A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication.

The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial transcription, and respiratory chain function. Finally, we present evidence that the HMG2 domain plays the leading role in TFAM species-specificity, thus indicating a potential pathway for TFAM-mtDNA evolutionary co-adaptations.

ExoU Induces Lung Endothelial Cell Damage and Activates Pro-Inflammatory Caspase-1 during Pseudomonas aeruginosa Infection.

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A2 (PLA2) that hydrolyses host cell membranes, and P. aeruginosa strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized P. aeruginosa strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA2-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA2-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with P. aeruginosa strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA2 activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from P. aeruginosa-infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during P. aeruginosa infection that may inhibit endothelial barrier and inflammatory functions.

Ablation of PDE4B protects from Pseudomonas aeruginosa-induced acute lung injury in mice by ameliorating the cytostorm and associated hypothermia.

Pseudomonas aeruginosa is a frequent cause of hospital-acquired lung infections characterized by hyperinflammation, antibiotic resistance, and high morbidity/mortality. Here, we show that the genetic ablation of one cAMP-phosphodiesterase 4 subtype, PDE4B, is sufficient to protect mice from acute lung injury induced by P aeruginosa infection as it reduces pulmonary and systemic levels of pro-inflammatory cytokines, as well as pulmonary vascular leakage and mortality. Surprisingly, despite dampening immune responses, bacterial clearance in the lungs of PDE4B-KO mice is significantly improved compared to WT controls. In wildtypes, P aeruginosa-infection produces high systemic levels of several cytokines, including TNF-α, IL-1ß, and IL-6, that act as cryogens and render the animals hypothermic. This, in turn, diminishes their ability to clear the bacteria. Ablation of PDE4B curbs both the initial production of acute response cytokines, including TNF-α and IL-1ß, as well as their downstream signaling, specifically the induction of the secondary-response cytokine IL-6. This synergistic action protects PDE4B-KO mice from the deleterious effects of the P aeruginosa-induced cytostorm, while concurrently improving bacterial clearance, rather than being immunosuppressive. These benefits of PDE4B ablation are in contrast to the effects resulting from treatment with PAN-PDE4 inhibitors, which have been shown to increase bacterial burden and dissemination. Thus, PDE4B represents a promising therapeutic target in settings of P aeruginosa lung infections.

Exoenzyme Y induces extracellular active caspase-7 accumulation independent from apoptosis: modulation of transmissible cytotoxicity.

Caspase-3 and -7 are executioner caspases whose enzymatic activity is necessary to complete apoptotic cell death. Here, we questioned whether endothelial cell infection leads to caspase-3/7-mediated cell death. Pulmonary microvascular endothelial cells (PMVECs) were infected with Pseudomonas aeruginosa (PA103). PA103 caused cell swelling with a granular appearance, paralleled by intracellular caspase-3/7 activation and cell death. In contrast, PMVEC infection with ExoY+ (PA103 ΔexoUexoT::Tc pUCPexoY) caused cell rounding, but it did not activate intracellular caspase-3/7 and it did not cause cell death. However, ExoY+ led to a time-dependent accumulation of active caspase-7, but not caspase-3, in the supernatant, independent of apoptosis. To study the function of extracellular caspase-7, caspase-7- and caspase-3-deficient PMVECs were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. Caspase-7 activity was significantly reduced in supernatants from infected caspase-7-deficient cells but was unchanged in supernatants from infected caspase-3 deficient cells, indicating an uncoupling in the mechanism of activation of these two enzymes. Because ExoY+ leads to the release of heat stable amyloid cytotoxins that are responsible for transmissible cytotoxicity, we next questioned whether caspase-7 contributes to the severity of this process. Supernatants obtained from infected caspase-7-deficient cells displayed significantly reduced transmissible cytotoxicity when compared with supernatants from infected wild-type controls, illustrating an essential role for caspase-7 in promoting the potency of transmissible cytotoxicity. Thus, we report a mechanism whereby ExoY+ infection induces active caspase-7 accumulation in the extracellular space, independent of both caspase-3 and cell death, where it modulates ExoY+-induced transmissible cytotoxicity.

cAMP signaling primes lung endothelial cells to activate caspase-1 during Pseudomonas aeruginosa infection.

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.

Elimination of Mitochondrial DNA from Mammalian Cells.

To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC). Support protocols detail approaches for (1) genotyping ρ° cells of human, mouse, and rat origin by PCR; (2) quantitation of mtDNA by quantitative PCR (qPCR); and (3) preparation of calibrator plasmids for mtDNA quantitation. © 2018 by John Wiley & Sons, Inc.

Correction: Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

[This corrects the article DOI: 10.1371/journal.pone.0152705.].

Methods for Efficient Elimination of Mitochondrial DNA from Cultured Cells.

Here, we document that persistent mitochondria DNA (mtDNA) damage due to mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase as well as mitochondrial overexpression of bacterial Exonuclease III or Herpes Simplex Virus protein UL12.5M185 can induce a complete loss of mtDNA (ρ0 phenotype) without compromising the viability of cells cultured in media supplemented with uridine and pyruvate. Furthermore, we use these observations to develop rapid, sequence-independent methods for the elimination of mtDNA, and demonstrate utility of these methods for generating ρ0 cells of human, mouse and rat origin. We also demonstrate that ρ0 cells generated by each of these three methods can serve as recipients of mtDNA in fusions with enucleated cells.

Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number.

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.

The efficiency of the translesion synthesis across abasic sites by mitochondrial DNA polymerase is low in mitochondria of 3T3 cells.

Translesion synthesis by specialized DNA polymerases is an important strategy for mitigating DNA damage that cannot be otherwise repaired either due to the chemical nature of the lesion. Apurinic/Apyrimidinic (abasic, AP) sites represent a block to both transcription and replication, and are normally repaired by the base excision repair (BER) pathway. However, when the number of abasic sites exceeds BER capacity, mitochondrial DNA is targeted for degradation. Here, we used two uracil-N-glycosylase (UNG1) mutants, Y147A or N204D, to generate AP sites directly in the mtDNA of NIH3T3 cells in vivo at sites normally occupied by T or C residues, respectively, and to study repair of these lesions in their native context. We conclude that mitochondrial DNA polymerase γ (Pol γ) is capable of translesion synthesis across AP sites in mitochondria of the NIH3T3 cells, and obeys the A-rule. However, in our system, base excision repair (BER) and mtDNA degradation occur more frequently than translesion bypass of AP sites.

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions.

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.

Naturally occurring mutations in the canine CFTR gene.

Naturally occurring cystic fibrosis (CF)-causing mutations in the CFTR gene have not been identified in any nonhuman animal species. Since domestic dogs are known to develop medical conditions associated with atypical CF in humans (e.g., bronchiectasis and pancreatitis), we hypothesized that dogs with these disorders likely have a higher expression rate of CFTR mutations than the at-large population. Temporal temperature-gradient gel electrophoresis (TTGE) was used to screen canine CFTR in 400 animals: 203 dogs diagnosed with pancreatitis, 23 dogs diagnosed with bronchiectasis, and 174 dogs admitted to clinics for any illness (at-large dogs). Twenty-eight dogs were identified with one of four CFTR missense mutations. P1281T and P1464H mutations occur in relatively unconserved residues. R1456W is analogous to the human R1453W mutation, which has approximately 20% of normal CFTR function and is associated with pancreatitis and panbronchiolitis. R812W disrupts a highly conserved protein kinase A recognition site within the regulatory domain. We conclude that naturally occurring CFTR mutations are relatively common in domestic dogs and can be detected with TTGE. No substantive differences in mutation frequency were observed between the at-large, pancreatitis, and bronchiectasis dogs.

Fluid secretion by submucosal glands of the tracheobronchial airways.

Submucosal glands of the tracheobronchial airways provide the important functions of secreting mucins, antimicrobial substances, and fluid. This review focuses on the ionic mechanism and regulation of gland fluid secretion and examines the possible role of gland dysfunction in the lethal disease cystic fibrosis (CF). The fluid component of gland secretion is driven by the active transepithelial secretion of both Cl(-) and HCO(3)(-) by serous cells. Gland fluid secretion is neurally regulated with acetylcholine, substance P, and vasoactive intestinal peptide (VIP) playing prominent roles. The cystic fibrosis transmembrane conductance regulator (CFTR) is present in the apical membrane of gland serous cells and mediates the VIP-induced component of liquid secretion whereas the muscarinic component of liquid secretion appears to be at least partially CFTR-independent. Loss of CFTR function, which occurs in CF disease, reduces the capacity of glands to secrete fluid but not mucins. The possible links between the loss of fluid secretion capability and the complex airway pathology of CF are discussed.

Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates.

BACKGROUND: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. RESULTS: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. CONCLUSION: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.

Helicobacter pylori thioredoxin is an arginase chaperone and guardian against oxidative and nitrosative stresses.

The gastric human pathogen Helicobacter pylori faces formidable challenges in the stomach including reactive oxygen and nitrogen intermediates. Here we demonstrate that arginase activity, which inhibits host nitric oxide production, is post-translationally stimulated by H. pylori thioredoxin (Trx) 1 but not the homologous Trx2. Trx1 has chaperone activity that renatures urea- or heat-denatured arginase back to the catalytically active state. Most reactive oxygen and nitrogen intermediates inhibit arginase activity; this damage is reversed by Trx1, but not Trx2. Trx1 and arginase equip H. pylori with a "renox guardian" to overcome abundant nitrosative and oxidative stresses encountered during the persistence of the bacterium in the hostile gastric environment.