Dataset Information

TET2_x001E_mediated PTPN6 demethylation drives post_x001E_burn immune paralysis and AAV_x001E_CRISPRi targeting PTPN6 improves secondary bloodstream infection outcome

ABSTRACT: Background Severe burn injury often leads to a state of immune paralysis, which markedly increases the risk of death from secondary bloodstream infection. However, the molecular mechanisms underlying post_x001E_burn immune paralysis, especially why it worsens infectious outcomes, remain poorly understood. This study aimed to elucidate the epigenetic regulatory mechanisms of post_x001E_burn immune paralysis and to evaluate the therapeutic potential of targeted intervention for secondary bloodstream infection. Methods A 30% total body surface area full_x001E_thickness (third_x001E_degree) burn model was established in C57BL/6J mice. Secondary bloodstream infection was induced by intravenous injection of Klebsiella pneumoniae or Pseudomonas aeruginosa on day 14 post_x001E_burn. Immune paralysis was assessed by monocyte phagocytosis, T_x001E_cell proliferation and IFN_x001E_γ secretion, and immune cell subset analysis. For mechanistic investigation, transcriptome sequencing was performed to identify key molecules, followed by bisulfite sequencing, molecular interaction assays, and in vivo CRISPRi/a_x001E_mediated knockdown or overexpression to delineate the epigenetic regulation and function. Finally, AAV_x001E_CRISPRi was used to knock down the target molecule in vivo, and its effects on immune paralysis and secondary infection outcomes were evaluated. Twenty_x001E_four severe burn patients and ten healthy volunteers were enrolled for clinical validation. Results Burn mice exhibited typical immune paralysis, characterized by decreased macrophage phagocytosis, impaired T_x001E_cell proliferation and IFN_x001E_γ secretion, increased M2/Treg ratios, and reduced pro_x001E_inflammatory cytokine levels. Mechanistically, PTPN6 was markedly up_x001E_regulated after burn and drove immune paralysis by suppressing the NF_x001E_κB and MAPK signaling pathways. Further investigation revealed that TET2 directly bound to the PTPN6 promoter and mediated its demethylation, leading to PTPN6 overexpression. In vivo knockdown of PTPN6 effectively alleviated immune paralysis, reduced bacterial load, attenuated multi_x001E_organ damage, and improved 14_x001E_day survival in burn mice (P < 0.01). Clinical sample analysis confirmed PTPN6 promoter demethylation, up_x001E_regulation of the TET2/PTPN6 axis, and immune dysfunction in peripheral blood of burn patients. Conclusions The key mechanism driving post_x001E_burn immune paralysis is “TET2_x001E_mediated PTPN6 promoter demethylation → PTPN6 up_x001E_regulation → suppression of NF_x001E_κB/MAPK pathways”. AAV_x001E_CRISPRi_x001E_mediated knockdown of PTPN6 effectively reverses immune paralysis and significantly improves the outcome of secondary bloodstream infection. This work provides a novel molecular basis for understanding post_x001E_burn immune dysfunction and identifies a potential therapeutic target for clinical intervention.

ORGANISM(S): Mus musculus

PROVIDER: GSE327575 | GEO | 2026/04/30

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:Deficiency in the protein-tyrosine phosphatase SHP1/PTPN6 is linked with hematological malignancies and chronic inflammatory diseases. Here we exploited the embryonic and larval stages of zebrafish as an animal model to study ptpn6 function in the sole context of innate immunity. We show that ptpn6 knockdown induces a spontaneous inflammation-associated phenotype at the late larval stage, which was microbe-independent and enhanced instead of suppressed by glucocorticoids. When challenged with Salmonella typhimurium or Mycobacterium marinum at earlier stages of development, the innate immune system was hyperactivated to a contra-productive level that impaired the control of these pathogenic bacteria. These results demonstrate the crucial regulatory function of ptpn6 in preventing host-detrimental effects of inflammation by imposing a tight control over the level of innate immune response activation. This microarray study was designed to determine the effect of morpholino knockdown of the ptpn6 gene on the innate immune response of zebrafish embryos during infection with Salmonella typhimurium. Three independent infection experiments were performed using mixed egg clutches of wild type zebrafish, which were a cross of the AB and TL strains. Each experiment consisted of the following four treatment groups: (1) uninfected control embryos, (2) S. typhimurium-infected control embryos, (3) uninfected ptpn6 knockdown embryos, and (4) S. typhimurium-infected ptpn6 knockdown embryos. Knockdown of ptpn6 was performed by micro-injecting embryos at the 1-2 cell stage with the ptpn6 morpholino, and control embryos were mock-injected with buffer. Embryos were grown at 28.5M-bM-^@M-^S30M-BM-0C in egg water and manually dechorionated at 24 hours post fertilization (hpf). Subsequently, embryos were infected at 28 hpf by micro-injecting 200-250 colony forming units (CFU) of S. typhimurium SL1027 bacteria into the caudal vein, or were mock-injected with buffer as a control. After injections embryos were transferred into fresh egg water and incubated for 8 h at 28M-BM-0C. After the incubation period, pools of 15-20 embryos per treatment group were snap-frozen in liquid nitrogen and RNA was isolated for microarray analysis. All treatment groups were analyzed using a common reference approach.

Project description:Type 2 Diabetes, obesity and metabolic syndrome are pathologies impacting a large population worldwide where insulin resistance plays a central role. These pathologies are usually associated to a dysregulation of insulin secretion leading to a chronic exposure of the tissues to high insulin levels (i.e. hyperinsulinemia) what diminishes the concentration of key downstream elements causing insulin resistance. The complexity of the study of insulin resistance relies on the heterogeneity of the metabolic states where itM-bM-^@M-^Ys observed. In consequence, animal models for the study of insulin resistance, can not completely recapitulate the metabolic status of insulin resistant humans, what is translated in contradictory observations. To contribute to the understanding of the mechanisms triggering insulin resistance we have developed a zebrafish model to study insulin metabolism and its associated disorders. Zebrafish embryos appeared to be sensitive to human recombinant insulin, becoming insulin resistant when exposed to a high dose of the hormone, as confirmed by glucose measurements. Moreover RNAseq-based transcriptomic profiling of these embryos revealed a strong down regulation of a number of immune relevant genes as a consequences of the exposure to hyperinsulinemia. Interestingly, as an exception, the negative immune modulator ptpn6 appeared to be up regulated in insulin resistant embryos. Knockdown of ptpn6 showed to counteract the observed down regulation of the immune system and insulin signalling pathways effects at the transcriptional level caused by hyperinsulinemia. These results show that ptpn6 is a mediator of the metabolic switch between insulin sensitive and insulin resistant states. Our zebrafish model for hyperinsulinemia has therefore demonstrated it suitability to discover novel regulators of insulin resistance. In addition, our data will be very useful to further study the function of immunological determinants in a non-obese model system. 16 samples in total were analyzed. 4 replicates from control samples (injected with PBS) and 4 replicates of insulin injected samples at 0.5 hpi and 4 hpi. In each sample 10 embryos were pooled.

Dataset Information

TET2_x001E_mediated PTPN6 demethylation drives post_x001E_burn immune paralysis and AAV_x001E_CRISPRi targeting PTPN6 improves secondary bloodstream infection outcome

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets