Celastrol mitigates inflammation in sepsis by inhibiting the PKM2-dependent Warburg effect

Background: Sepsis involves life-threatening organ dysfunction and is caused by a dysregulated host response to infection. No specific therapies against sepsis have been reported. Celastrol(Cel) is a natural anti-inflammatory compound that shows potential against systemic inflammatory diseases. This study aimed to investigate the pharmacological activity and molecular mechanism of Cel in models of endotoxemia and sepsis.Methods: We evaluated the anti-inflammatory efficacy of Cel against endotoxemia and sepsis in mice and macrophage cultures treated with lipopolysaccharide(LPS). We screened for potential protein targets of Cel using activity-based protein profiling(ABPP). Potential targets were validated using biophysical methods such as cellular thermal shift assays(CETSA) and surface plasmon resonance(SPR). Residues involved in Cel binding to target proteins were identified through point mutagenesis, and the functional effects of such binding were explored through gene knockdown.Results: Cel protected mice from lethal endotoxemia and improved their survival with sepsis, and it significantly decreased the levels of pro-inflammatory cytokines in mice and macrophages treated with LPS(P <0.05). Cel bound to Cys424 of pyruvate kinase M2(PKM2), inhibiting the enzyme and thereby suppressing aerobic glycolysis(Warburg effect). Cel also bound to Cys106 in high mobility group box 1(HMGB1) protein, reducing the secretion of inflammatory cytokine interleukin(IL)-1β. Cel bound to the Cys residues in lactate dehydrogenase A(LDHA).Conclusions: Cel inhibits inflammation and the Warburg effect in sepsis via targeting PKM2 and HMGB1 protein.

A metabolite from commensal Candida albicans enhances the bactericidal activity of macrophages and protects against sepsis

The gut microbiome is recognized as a key modulator of sepsis development.However,the contribution of the gut mycobiome to sepsis development is still not fully understood.Here,we demonstrated that the level of Candida albicans was markedly decreased in patients with bacterial sepsis,and the supernatant of Candida albicans culture significantly decreased the bacterial load and improved sepsis symptoms in both cecum ligation and puncture(CLP)-challenged mice and Escherichia coli-challenged pigs.Integrative metabolomics and the genetic engineering of fungi revealed that Candida albicans-derived phenylpyruvate(PPA)enhanced the bactericidal activity of macrophages and reduced organ damage during sepsis.Mechanistically,PPA directly binds to sirtuin 2(SIRT2)and increases reactive oxygen species(ROS)production for eventual bacterial clearance.Importantly,PPA enhanced the bacterial clearance capacity of macrophages in sepsis patients and was inversely correlated with the severity of sepsis in patients.Our findings highlight the crucial contribution of commensal fungi to bacterial disease modulation and expand our understanding of the host-mycobiome interaction during sepsis development.

High-fat diet impairs gut barrier through intestinal microbiota-derived reactive oxygen species

Gut barrier disruption is a key event in bridging gut microbiota dysbiosis and high-fat diet(HFD)-associated metabolic disorders.However,the underlying mechanism remains elusive.In the present study,by comparing HFD-and normal diet(ND)-treated mice,we found that the HFD instantly altered the composition of the gut microbiota and subsequently damaged the integrity of the gut barrier.Metagenomic sequencing revealed that the HFD upregulates gut microbial functions related to redox reactions,as confirmed by the increased reactive oxygen species(ROS)levels in fecal microbiota incubation in vitro and in the lumen,which were detected using in vivo fluorescence imaging.This microbial ROS-producing capability induced by HFD can be transferred through fecal microbiota transplantation(FMT)into germ-free(GF)mice,downregulating the gut barrier tight junctions.Similarly,mono-colonizing GF mice with an Enterococcus strain excelled in ROS production,damaged the gut barrier,induced mitochondrial malfunction and apoptosis of the intestinal epithelial cells,and exacerbated fatty liver,compared with other low-ROS-producing Enterococcus strains.Oral administration of recombinant high-stability-superoxide dismutase(SOD)significantly reduced intestinal ROS,protected the gut barrier,and improved fatty liver against the HFD.In conclusion,our study suggests that extracellular ROS derived from gut microbiota play a pivotal role in HFD-induced gut barrier disruption and is a potential therapeutic target for HFD-associated metabolic diseases.

Mapk7 deletion in chondrocytes causes vertebral defects by reducing MEF2C/PTEN/AKT signaling

Mutation of the MAPK7 gene was related to human scoliosis.Mapk7 regulated the development of limb bones and skulls in mice.However,the role of MAPK7 in vertebral development is still unclear.In this study,we constructed Col2a1-cre;Mapk7 f/f transgenic mouse model to delete Mapk7 in cartilage,which displayed kyphosis and osteopenia.Mechanistically,Mapk7 loss decreased MEF2C expression and thus activated PTEN to oppose PI3K/AKT signaling in vertebral growth plate chondrocytes,which impaired chondrocyte hypertrophy and attenuated vertebral ossification.In vivo,systemic pharmacological activation of AKT rescued impaired chondrocyte hypertrophy and alleviated mouse vertebral defects caused by Mapk7 deficiency.Our study firstly clarified the mechanism by which MAPK7 was involved in vertebral development,which might contribute to understanding the pathology of spinal deformity and provide a basis for the treatment of developmental disorders of the spine.

Targeting complement hyperactivation:a novel therapeutic approach for severe pneumonia induced by influenza virus/staphylococcus aureus coinfection

Dear Editor,Infections by the influenza virus are a significant and widespread global health threat,as these infections have an annual death toll ranging from 290,000 to 650,000.1 A significant proportion of these fatalities are attributed to secondary bacterial pneumonia,a severe complication commonly caused by ubiquitous respiratory pathogens such as Staphylococcus aureus(S.aureus).2 Of particular concern is the increased morbidity and mortality rates in individuals infected simultaneously with influenza virus and methicillin-resistant S.aureus(MRSA).3 The concurrent presence of bacteria and influenza virus usually causes acute respiratory distress syndrome(ARDS),which is associated with acute lung injury(ALI),severe lung tissue edema,and widespread inflammation.Nevertheless,determining the complex mechanisms underlying the synergistic interplay will require further investigation with a suitable coinfection mouse model.In our previous study,we utilized different sequential coinfections at various time points to model influenza A virus and MRSA coinfection.

The gut microbial metabolite, 3,4-dihydroxyphenylpropionic acid, alleviates hepatic ischemia/reperfusion injury via mitigation of macrophage pro-inflammatory activity in mice

Hepatic ischemia/reperfusion injury(HIRI) is a serious complication that occurs following shock and/or liver surgery. Gut microbiota and their metabolites are key upstream modulators of development of liver injury. Herein, we investigated the potential contribution of gut microbes to HIRI.Ischemia/reperfusion surgery was performed to establish a murine model of HIRI. 16 S r RNA gene sequencing and metabolomics were used for microbial analysis. Transcriptomics and proteomics analysis were employed to study the host cell responses. Our results establish HIRI was significantly increased when surgery occurred in the evening(ZT12, 20:00) when compared with the morning(ZT0, 08:00);however, antibiotic pretreatment reduced this diurnal variation. The abundance of a microbial metabolite3,4-dihydroxyphenylpropionic acid was significantly higher in ZT0 when compared with ZT12 in the gut and this compound significantly protected mice against HIRI. Furthermore, 3,4-dihydroxyphenylpropionic acid suppressed the macrophage pro-inflammatory response in vivo and in vitro. This metabolite inhibits histone deacetylase activity by reducing its phosphorylation. Histone deacetylase inhibition suppressed macrophage pro-inflammatory activation and diminished the diurnal variation of HIRI. Our findings revealed a novel protective microbial metabolite against HIRI in mice. The potential underlying mechanism was at least in part, via 3,4-dihydroxyphenylpropionic acid-dependent immune regulation and histone deacetylase(HDAC) inhibition in macrophages.

clusterProfiler 4.0:A universal enrichment tool for interpreting omics data

Functional enrichment analysis is pivotal for interpreting highthroughput omics data in life science.It is crucial for this type of tool to use the latest annotation databases for as many organisms as possible.To meet these requirements,we present here an updated version of our popular Bioconductor package,clusterProfiler 4.0.This package has been enhanced considerably compared with its original version published 9 years ago.The new version provides a universal interface for functional enrichment analysis in thousands of organisms based on internally supported ontologies and pathways as well as annotation data provided by users or derived from online databases.It also extends the dplyr and ggplot2 packages to offer tidy interfaces for data operation and visualization.Other new features include gene set enrichment analysis and comparison of enrichment results from multiple gene lists.We anticipate that clusterProfiler 4.0 will be applied to a wide range of scenarios across diverse organisms.

Identification of heptapeptides targeting a lethal bacterial strain in septic mice through an integrative approach

Effectively killing pathogenic bacteria is key for the treatment of sepsis.Although various anti-infective drugs have been used for the treatment of sepsis,the therapeutic effect is largely limited by the lack of a specific bacterium-targeting delivery system.This study aimed to develop antibacterial peptides that specifically target pathogenic bacteria for the treatment of sepsis.The lethal bacterial strain Escherichia coli MSI001 was isolated from mice of a cecal ligation and puncture(CLP)model and was used as a target to screen bacterial binding heptapeptides through an integrative bioinformatics approach based on phage display technology and high-throughput sequencing(HTS).Heptapeptides binding to E.coli MSI001 with high affinity were acquired after normalization by the heptapeptide frequency of the library.A representative heptapeptide VTKLGSL(VTK)was selected for fusion with the antibacterial peptide LL-37 to construct the specific-targeting antibacterial peptide VTK-LL37.We found that,in comparison with LL37,VTK-LL37 showed prominent bacteriostatic activity and an inhibitive effect on biofilm formation in vitro.In vivo experiments demonstrated that VTK-LL37 significantly inhibited bacterial growth,reduced HMGB1 expression,alleviated lesions of vital organs and improved the survival of mice subjected to CLP modeling.Furthermore,membrane DEGP and DEGQ were identified as VTKbinding proteins by proteomic methods.This study provides a novel strategy for targeted pathogen killing,which is helpful for the treatment of sepsis in the era of precise medicine.

Crosstalk between gut microbiota and sepsis

Sepsis is an overwhelming inflammatory response to microbial infection.Sepsis management remains a clinical challenge.The role of the gut microbiome in sepsis has gained some attention.Recent evidence has demonstrated that gut microbiota regulate host physiological homeostasis mediators,including the immune system,gut barrier function and disease susceptibility pathways.Therefore,maintenance or restoration of microbiota and metabolite composition might be a therapeutic or prophylactic target against critical illness.Fecal microbiota transplantation and supplementation of probiotics are microbiota-based treatment methods that are somewhat limited in terms of evidence-based efficacy.This review focuses on the importance of the crosstalk between the gastrointestinal ecosystem and sepsis to highlight novel microbiota-targeted therapies to improve the outcomes of sepsis treatment.

Rab20 is critical for bacterial lipoprotein tolerization-enhanced bactericidal activity in macrophages during bacterial infection

Bacterial cell wall component-induced tolerance represents an important protective mechanism during microbial infection.Tolerance induced by the TLR2 agonist bacterial lipoprotein(BLP)has been shown to attenuate the inflammatory response,and simultaneously to augment antimicrobial function,thereby conferring its protection against microbial sepsis.However,the underlying mechanism by which BLP tolerance augments bactericidal activity has not been fully elucidated.Here,we reported that the induction of BLP tolerance in murine macrophages upregulated the expression of Rab20,a membrane trafficking regulator,at both the mRNA and protein levels upon bacterial infection.The knockdown of Rab20 with Rab20 specific siRNA(siRab20)did not affect the phagocytosis of Escherichia coli(E.coli),but substantially impaired the intracellular killing of the ingested E.coli in BLP-tolerized macrophages.Furthermore,Rab20 was associated with GFP-E.coli containing phagosomes,and BLP tolerization resulted in the enhanced maturation of GFP-E.coli-containing phagosomes associated with Rab20 and strong lysosomal acidification.The knockdown of Rab20 substantially diminished lysosome acidification and disturbed the fusion of GFP-E.coli containing phagosomes with lysosomes in BLP-tolerized macrophages.These results demonstrate that Rab20 plays a critical role in BLP tolerization-induced augmentation of bactericidal activity via promoting phagosome maturation and the fusion of bacteria containing phagosomes with lysosomes.

Intestinal epithelial chemokine (C-C motif) ligand 7 overexpression protects against high fat diet-induced obesity and hepatic steatosis in mice

Background:We previously found that the intestinal epithelial chemokine(C-C motif)ligand 7(CCL7)plays an important role in the development of toxin-induced acute liver damage.The detailed effects of intestinal epithelial CCL7 on chronic diseases;however,are still unclear.Here,we aimed to investigate the impact of intestinal epithelial CCL7 overexpression on high-fat diet(HFD)-induced obesity and steatohepatitis in mice.Methods:Intestinal epithelial CCL7 overexpression(CCL7tgIEC)mice and their wild-type(WT)littermates were fed with normal chow or HFD for 16 weeks to induce obesity and non-alcoholic fatty liver disease.Body weight gain,as well as adipose tissue index were assessed.Liver injury was monitored by histological analysis and real time polymerase chain reaction.Gut microbial composition was analyzed by 16S rRNA gene sequencing.Results:We found that the CCL7tgIEC mice on a HFD had markedly decreased weight gain(8.9 vs.17.0 g,P<0.05)and a lower adipose tissue index that include mesenteric fat(1.0%vs.1.76%,P<0.05),gonadal fat(2.1%vs.6.1%,P<0.05),subcutaneous fat(1.0%vs.2.8%,P<0.05)compared to WT animals.HFD-induced glucose intolerance and insulin resistance were also significantly improved in CCL7tgIEC mice compared to WT.Furthermore,HFD-fed CCL7tgIEC mice displayed less hepatic lipid accumulation and lower expression of inflammatory factors than WT mice.16S rRNA gene sequencing demonstrated that CCL7 overexpression in intestinal epithelial cells improved HFD-induced gut microbial dysbiosis.Conclusions:Our study revealed that CCL7 overexpression in the intestinal epithelium protects mice against the progression of diet-induced obesity,hepatic steatosis,and enteric dysbiosis.

Irreversible electroporation plus allogenic Vγ9Vδ2 T cells enhances antitumor effect for locally advanced pancreatic cancer patients

Immunotherapy has limited efficacy against locally advanced pancreatic cancer(LAPC)due to the presence of an immunosuppressive microenvironment(ISM).Irreversible electroporation(IRE)can not only induce immunogenic cell death,but also alleviate immunosuppression.This study aimed to investigate the antitumor efficacy of IRE plus allogeneicγδT cells in LAPC patients.A total of 62 patients who met the eligibility criteria were enrolled in this trial,then randomized into two groups(A:n=30 and B:n=32).All patients received IRE therapy and after receiving IRE,the group A patients received at least two cycles ofγδT-cell infusion as one course continuously.Group A patients had better survival than group B patients(median OS:14.5 months vs.11 months;median PFS:11 months vs.8.5 months).Moreover,the group A patients treated with multiple courses ofγδT-cell infusion had longer OS(17 months)than those who received a single course(13.5 months).IRE combined with allogeneicγδT-cell infusion is a promising strategy to enhance the antitumor efficacy in LAPC patients,yielding extended survival benefits.

Enteric dysbiosis, gut barrier and liver disease

By employing the powerful animal model—Germ free mice and the development of the sequencing technology,gut microbiota nowadays has been recognized as a key pathophysiologic modulator for many diseases development,including liver injury progression.