Deletion of Interleukin-1ß Converting Enzyme Alters Mouse Cardiac Structure and Function.

Interleukin-1ß converting enzyme (ICE, caspase-1) is a thiol protease that cleaves the pro-inflammatory cytokine precursors of IL-1ß and IL-18 into active forms. Given the association between caspase-1 and cardiovascular pathology, we analyzed the hearts of ICE knockout (ICE KO) mice to test the hypothesis that caspase-1 plays a significant role in cardiac morphology and function. We characterized the histological and functional changes in the hearts of ICE KO mice compared to the Wild type. The cardiomyocytes from the neonatal ICE KO mice showed an impaired response to oxidative stress. Subsequently, the hearts from the ICE KO mice were hypertrophied, with a significant increase in the left ventricular and septal wall thickness and a greater LV mass/body weight ratio. The ICE KO mice hearts exhibited irregular myofibril arrangements and disruption of the cristae in the mitochondrial structure. Proapoptotic proteins that were significantly increased in the hearts of ICE KO versus the Wild type included pErk, pJNK, p53, Fas, Bax, and caspase 3. Further, the antiapoptotic proteins Bag-1 and Bcl-2 are activated in ICE KO hearts. Functionally, there was an increase in the left ventricular epicardial diameter and volume in ICE KO. In conclusion, our findings support the important role of caspase-1 in maintaining cardiac health; specifically, a significant decrease in caspase-1 is detrimental to the cardiovascular system.

Short-Term Ingestion of Essential Amino Acid Based Nutritional Supplements or Whey Protein Improves the Physical Function of Older Adults Independently of Gut Microbiome.

SCOPE: Dietary proteins and essential amino acids (EAAs) are the major nutritional supplements that support the growth and activity of gut microbes contributing to the wellbeing of their host. This study hypothesizes that daily supplementation of the diet with either EAAs or whey protein for 12 weeks would improve the gut microbiome of older adults. METHODS AND RESULTS: The stool samples are processed and subjected to Illumina-based 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing. In both groups, the most abundant families are found in order of relative abundance included: Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Rikenellaceae, Enterobacteriaceae, Oscillospiraceae, Tannerellaceae, and Akkermansiaceae, which indicate that these subjects are able to maintain a same healthy microbial diversity in their guts. A significant finding is a reduction of proinflammatory cytokine, interleukin-18 (IL-18) in the EAAs group. It also uses the standard 6-min walking test (6MWT) as a measure of cardiopulmonary fitness. At the end of the study, the subjects in the EAAs group perform significantly better in the 6MWT as compared to the whey group. CONCLUSION: It seems plausible that the improved physical performance and reduced proinflammatory cytokine, IL-18 seen in the EAAs group, are independent of changes in gut microbiota.

Proteomic analysis of P. gingivalis-Lipopolysaccharide induced neuroinflammation in SH-SY5Y and HMC3 cells.

Chronic periodontitis and its keystone pathogen, Porphyromonas gingivalis, have increasingly been linked with Alzheimer's disease (AD). However, P.gingivalis-lipopolysaccharide (LPS) mediated release of neuroinflammatory proteins contributes to AD remains underexplored. In this study, we utilized data-independent acquisition mass spectrometry to characterize P.gingivalis-LPS induced profile of differentially expressed proteins associated with the neuroinflammatory response in human neuroblastoma (SH-SY5Y) and human microglial (HMC3) cells. We reported a set of 124 proteins in SH-SY5Y cells and 96 proteins in HMC3 cells whose levels were significantly upregulated or downregulated by exposure to P. gingivalis-LPS. Our findings demonstrate that P. gingivalis-LPS contributed to the elevated expressions of dementia biomarkers and pro-inflammatory cytokines that include APP, Aß1-42, Aß1-40, T-Tau, p-Tau, VEGF, TGF-ß, IL-1ß, IL-6 and TNF-α through 2 distinct pathways of extracellular sensing by cell surface receptors and intracellular cytosolic receptors. Interestingly, intracellular signaling proteins activated with P. gingivalis-LPS transfection using Lipofectamine™ 2000 had significantly higher fold change protein expression compared to the extracellular signaling with P. gingivalis-LPS treatment. Additionally, we also explored P. gingivalis-LPS mediated activation of caspase-4 dependent non canonical inflammasome pathway in both SH-SY5Y and HMC3 cells. In summary, P. gingivalis-LPS induced neuroinflammatory protein expression in SH-SY5Y and HMC3 cells, provided insights into the specific inflammatory pathways underlying the potential link between P. gingivalis-LPS infection and the pathogenesis of Alzheimer's disease and related dementias.

Inhibitors of Rho/MRTF/SRF Transcription Pathway Regulate Mitochondrial Function.

RhoA-regulated gene transcription by serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factors (MRTFs) signaling pathway has emerged as a promising therapeutic target for pharmacological intervention in multiple diseases. Altered mitochondrial metabolism is one of the major hallmarks of cancer, therefore, this upregulation is a vulnerability that can be targeted with Rho/MRTF/SRF inhibitors. Recent advances identified a novel series of oxadiazole-thioether compounds that disrupt the SRF transcription, however, the direct molecular target of these compounds is unclear. Herein, we demonstrate the Rho/MRTF/SRF inhibition mechanism of CCG-203971 and CCG-232601 in normal cell lines of human lung fibroblasts and mouse myoblasts. Further studies investigated the role of these molecules in targeting mitochondrial function. We have shown that these molecules hyperacetylate histone H4K12 and H4K16 and regulate the genes involved in mitochondrial function and dynamics. These small molecule inhibitors regulate mitochondrial function as a compensatory mechanism by repressing oxidative phosphorylation and increasing glycolysis. Our data suggest that these CCG molecules are effective in inhibiting all the complexes of mitochondrial electron transport chains and further inducing oxidative stress. Therefore, our present findings highlight the therapeutic potential of CCG-203971 and CCG-232601, which may prove to be a promising approach to target aberrant bioenergetics.

Valine improves mitochondrial function and protects against oxidative stress.

Among the branched-chain amino acids, leucine and isoleucine have been well studied for their roles in improving mitochondrial function and reducing oxidative stress. However, role of valine in mitochondrial function regulation and oxidative stress management remains elusive. This study investigated valine effect on mitochondrial function and oxidative stress in vitro. Valine increased expression of genes involved in mitochondrial biogenesis and dynamics. It upregulates mitochondrial function at complexes I, II, and IV levels of electron transport chain. Flow cytometry studies revealed, valine reduced oxidative stress by significantly lowering mitochondrial reactive oxygen species and protein expression of 4-hydroxynonenal. Functional role of valine against oxidative stress was analyzed by XFe96 Analyzer. Valine sustained oxidative phosphorylation and improved ATP generation rates during oxidative stress. In conclusion, our findings shed more light on the critical function of valine in protecting mitochondrial function thereby preventing mitochondrial/cellular damage induced by oxidative stress.

P. gingivalis-LPS Induces Mitochondrial Dysfunction Mediated by Neuroinflammation through Oxidative Stress.

Porphyromonas gingivalis (P. gingivalis), a key pathogen in periodontitis, is associated with neuroinflammation. Periodontal disease increases with age; 70.1% of adults 65 years and older have periodontal problems. However, the P. gingivalis- lipopolysaccharide (LPS)induced mitochondrial dysfunction in neurodegenerative diseases remains elusive. In this study, we investigated the possible role of P. gingivalis-LPS in mitochondrial dysfunction during neurodegeneration. We found that P. gingivalis-LPS treatment activated toll-like receptor (TLR) 4 signaling and upregulated the expression of Alzheimer's disease-related dementia and neuroinflammatory markers. Furthermore, the LPS treatment significantly exacerbated the production of reactive oxygen species and reduced the mitochondrial membrane potential. Our study highlighted the pivotal role of P. gingivalis-LPS in the repression of serum response factor (SRF) and its co-factor p49/STRAP that regulate the actin cytoskeleton. The LPS treatment repressed the genes involved in mitochondrial function and biogenesis. P. gingivalis-LPS negatively altered oxidative phosphorylation and glycolysis and reduced total adenosine triphosphate (ATP) production. Additionally, it specifically altered the mitochondrial functions in complexes I, II, and IV of the mitochondrial electron transport chain. Thus, it is conceivable that P. gingivalis-LPS causes mitochondrial dysfunction through oxidative stress and inflammatory events in neurodegenerative diseases.

Differential plasma protein expression after ingestion of essential amino acid-based dietary supplement verses whey protein in low physical functioning older adults.

In a recent randomized, double-blind, placebo-controlled trial, we were able to demonstrate the superiority of a dietary supplement composed of essential amino acids (EAAs) over whey protein, in older adults with low physical function. In this paper, we describe the comparative plasma protein expression in the same subject groups of EAAs vs whey. The plasma proteomics data was generated using SOMA scan assay. A total of twenty proteins were found to be differentially expressed in both groups with a 1.5-fold change. Notably, five proteins showed a significantly higher fold change expression in the EAA group which included adenylate kinase isoenzyme 1, casein kinase II 2-alpha, Nascent polypeptide-associated complex subunit alpha, peroxiredoxin-1, and peroxiredoxin-6. These five proteins might have played a significant role in providing energy for the improved cardiac and muscle strength of older adults with LPF. On the other hand, fifteen proteins showed slightly lower fold change expression in the EAA group. Some of these 15 proteins regulate metabolism and were found to be associated with inflammation or other comorbidities. Gene Ontology (GO) enrichment analysis showed the association of these proteins with several biological processes. Furthermore, protein-protein interaction network analysis also showed distinct networks between upregulated and downregulated proteins. In conclusion, the important biological roles of the upregulated proteins plus better physical function of participants in the EAAs vs whey group demonstrated that EAAs have the potential to improve muscle strength and physical function in older adults. This study was registered with ClinicalTrials.gov: NCT03424265 "Nutritional interventions in heart failure."

Neutrophil inflammasomes sense the subcellular delivery route of translocated bacterial effectors and toxins.

In neutrophils, caspase-11 cleaves gasdermin D (GSDMD), causing pyroptosis to clear cytosol-invasive bacteria. In contrast, caspase-1 also cleaves GSDMD but seems to not cause pyroptosis. Here, we show that this pyroptosis-resistant caspase-1 activation is specifically programmed by the site of translocation of the detected microbial virulence factors. We find that pyrin and NLRC4 agonists do not trigger pyroptosis in neutrophils when they access the cytosol from endosomal compartment. In contrast, when the same ligands penetrate through the plasma membrane, they cause pyroptosis. Consistently, pyrin detects extracellular Yersinia pseudotuberculosis ΔyopM in neutrophils, driving caspase-1-GSDMD pyroptosis. This pyroptotic response drives PAD4-dependent H3 citrullination and results in extrusion of neutrophil extracellular traps (NETs). Our data indicate that caspase-1, GSDMD, or PAD4 deficiency renders mice more susceptible to Y. pseudotuberculosis ΔyopM infection. Therefore, neutrophils induce pyroptosis in response to caspase-1-activating inflammasomes triggered by extracellular bacterial pathogens, but after they phagocytose pathogens, they are programmed to forego pyroptosis.

Rho/SRF Inhibitor Modulates Mitochondrial Functions.

CCG-1423 is a Rho A pathway inhibitor that has been reported to inhibit Rho/SRF-mediated transcriptional regulation. Serum response factor and its cofactors, which include ternary complex factors and myocardin-related transcription factors, regulate various cellular functions. In this study, we observed that CCG-1423 modulates the mitochondrial functions. The effect of this small molecule drug was determined by measuring mitochondrial function using an XFe96 Analyzer and an Oxygraph 2k (O2k) high-resolution respirometer. CCG-1423 treatment significantly reduced oxidative phosphorylation in a dose-dependent manner. However, CCG-1423 increased the glycolytic rate. We also observed that histone 4 at lysine-16 underwent hyperacetylation with the treatment of this drug. Immunolabeling with F-actin and MitoTracker revealed the alteration in the actin cytoskeleton and mitochondria. Taken together, our findings highlight a critical role of CCG-1423 in inhibiting the transcription of SRF/p49 and PGC-1α, ß, resulting in the downregulation of mitochondrial genes, leading to the repression of mitochondrial oxidative phosphorylation and overall ATP reduction. This study provides a better understanding of the effects of CCG-1423 on mitochondria, which may be useful for the assessment of the potential clinical application of CCG-1423 and its derivatives.

Shigella OspC3 suppresses murine cytosolic LPS sensing.

Shigella flexneri, a cytosol-invasive gram-negative pathogen, deploys an array of type III-secreted effector proteins to evade host cell defenses. Caspase-11 and its human ortholog caspase-4 detect cytosolic lipopolysaccharide (LPS) and trigger gasdermin D-mediated pyroptosis to eliminate intra-cytoplasmic bacterial threats. However, the role of caspase-11 in combating S. flexneri is unclear. The Shigella T3SS effector OspC3 reportedly suppresses cytosolic LPS sensing by inhibiting caspase-4 but not caspase-11 activity. Surprisingly, we found that S. flexneri also uses OspC3 to inhibit murine caspase-11 activity. Mechanistically, we found that OspC3 binds only to primed caspase-11. Importantly, we demonstrate that S. flexneri employs OspC3 to prevent caspase-11-mediated pyroptosis in neutrophils, enabling bacteria to disseminate and evade clearance following intraperitoneal challenge. In contrast, S. flexneri lacking OspC3 is attenuated in a caspase-11- and gasdermin D-dependent fashion. Overall, our study reveals that OspC3 suppresses cytosolic LPS detection in a broad array of mammals.

Caspase-11 Non-canonical Inflammasomes in the Lung.

The airway epithelium and underlying innate immune cells comprise the first line of host defense in the lung. They recognize pathogen-associated molecular patterns (PAMPs) using membrane-bound receptors, as well as cytosolic receptors such as inflammasomes. Inflammasomes activate inflammatory caspases, which in turn process and release the inflammatory cytokines IL-1ß and IL-18. Additionally, inflammasomes trigger a form of lytic cell death termed pyroptosis. One of the most important inflammasomes at the host-pathogen interface is the non-canonical caspase-11 inflammasome that responds to LPS in the cytosol. Caspase-11 is important in defense against Gram-negative pathogens, and can drive inflammatory diseases such as LPS-induced sepsis. However, pathogens can employ evasive strategies to minimize or evade host caspase-11 detection. In this review, we present a comprehensive overview of the function of the non-canonical caspase-11 inflammasome in sensing of cytosolic LPS, and its mechanism of action with particular emphasis in the role of caspase-11 in the lung. We also explore some of the strategies pathogens use to evade caspase-11.

Neutrophil Caspase-11 Is Essential to Defend against a Cytosol-Invasive Bacterium.

Either caspase-1 or caspase-11 can cleave gasdermin D to cause pyroptosis, eliminating intracellular replication niches. We previously showed that macrophages detect Burkholderia thailandensis via NLRC4, triggering the release of interleukin (IL)-18 and driving an essential interferon (IFN)-γ response that primes caspase-11. We now identify the IFN-γ-producing cells as a mixture of natural killer (NK) and T cells. Although both caspase-1 and caspase-11 can cleave gasdermin D in macrophages and neutrophils, we find that NLRC4-activated caspase-1 triggers pyroptosis in macrophages, but this pathway does not trigger pyroptosis in neutrophils. In contrast, caspase-11 triggers pyroptosis in both macrophages and neutrophils. This translates to an absolute requirement for caspase-11 in neutrophils during B. thailandensis infection in mice. We present an example of inflammasome sensors causing diverging outcomes in different cell types. Thus, cell fates are dictated not simply by the pathogen or inflammasome, but also by how the cell is wired to respond to detection events.