ABSTRACT
Background: As a damage-associated molecular pattern, the myeloid-related protein 8/14 (MRP8/14) heterodimer mediates various inflammatory diseases, such as sepsis. However, how MRP8/14 promotes lung injury by regulating the inflammatory response during endotoxemia remains largely unknown. This study aims at illuminating the pathological functions of MRP8/14 in endotoxemia. Methods: An endotoxemic model was prepared with wild-type and myeloid cell-specific Mrp8 deletion (Mrp8ΔMC) mice for evaluating plasma cytokine levels. Lung injury was evaluated by hematoxylin and eosin (H&E) staining, injury scoring and wet-to-dry weight (W/D) ratio. The dynamic profile of interferon γ (IFNγ)-inducible protein 10 (IP-10) mRNA expression induced by macrophage MRP8/14 was determined by quantitative real-time polymerase chain reaction (qPCR). Immunoblotting was used to evaluate the increase in IP-10 level induced by activation of the JAK-STAT signaling pathway. Luciferase reporter assay was performed to detect the involvement of IRF7 in Ip-10 gene transcription. In vivo air pouch experiments were performed to determine the biological function of IP-10 induced by MRP8/14. Results: Experiments with Mrp8ΔMC mice showed that MRP8/14 promoted the production of cytokines, including IP-10, in the bronchoalveolar lavage fluid (BALF) and lung injury in endotoxic mice. The result of qPCR showed sustained expression of Ip-10 mRNA in macrophages after treatment with MRP8/14 for 12 h. Neutralization experiments showed that the MRP8/14-induced Ip-10 expression in RAW264.7 cells was mediated by extracellular IFNß. Western blotting with phosphorylation-specific antibodies showed that the JAK1/TYK2-STAT1 signaling pathway was activated in MRP8/14-treated RAW264.7 cells, leading to the upregulation of Ip-10 gene expression. IRF7 was further identified as a downstream regulator of the JAK-STAT pathway that mediated Ip-10 gene expression in macrophages treated with MRP8/14. In vivo air pouch experiments confirmed that the IFNß-JAK1/TYK2-STAT1-IRF7 pathway was required for chemokine (C-X-C motif) receptor 3 (CXCR3)+ T lymphocyte migration, which promoted lung injury in the context of endotoxemia. Conclusions: In summary, our study demonstrates that MRP8/14 induces sustained production of IP-10 via the IFNß-JAK1/TYK2-STAT1-IRF7 pathway to attract CXCR3+ T lymphocytes into lung tissues and ultimately results in lung injury by an excessive inflammatory response in the context of endotoxemia.
ABSTRACT
Toll-like receptor 3 (TLR3), one pattern recognition receptor activated by viral and endogenous RNA, has been recently reported to regulate ischemia/reperfusion (I/R) injury in various organs. However, the role of TLR3 in the development of intestinal I/R injury remains unclear. The aim of this study is to evaluate the effects of extracellular RNAs/TLR3 signaling in intestinal I/R injury. An intestinal I/R injury model was established with superior mesenteric artery occlusion both in wild-type and TLR3 knockout (KO, -/-) mice, and MODE-K cells were subjected to hypoxia/reoxygenation (H/R) to mimic the I/R model in vivo. Extracellular RNAs (exRNAs), especially double-stranded RNAs (dsRNAs) co-localized with TLR3, were significantly increased both in vitro and in vivo. Compared with wild-type mice, TLR3 knockout obviously attenuated intestinal I/R injury. Both TLR3/dsRNA complex inhibitor and TLR3 siRNA administration reduced TLR3 expressions and subsequently inhibited intestinal inflammatory cytokine production and apoptosis. In conclusion, exRNAs/TLR3 signaling is a key mechanism that regulates intestinal I/R injury in adult mice, and the TLR3/dsRNA complex inhibitor can be an effective approach for attenuating intestinal I/R-induced inflammatory response and apoptosis.
Subject(s)
Reperfusion Injury , Toll-Like Receptor 3 , Mice , Animals , Toll-Like Receptor 3/genetics , Mice, Knockout , RNA , Reperfusion Injury/genetics , Reperfusion Injury/metabolism , Reperfusion , IschemiaABSTRACT
INTRODUCTION: Acute lung injury (ALI) is a major cause of morbidity and mortality after intestinal ischaemia/reperfusion (I/R). The gut microbiota and its metabolic byproducts act as important modulators of the gut-lung axis. This study aimed to define the role of succinate, a key microbiota metabolite, in intestinal I/R-induced ALI progression. METHODS: Gut and lung microbiota of mice subjected to intestinal I/R were analysed using 16S rRNA gene sequencing. Succinate level alterations were measured in germ-free mice or conventional mice treated with antibiotics. Succinate-induced alveolar macrophage polarisation and its effects on alveolar epithelial apoptosis were evaluated in succinate receptor 1 (Sucnr1)-deficient mice and in murine alveolar macrophages transfected with Sucnr1-short interfering RNA. Succinate levels were measured in patients undergoing cardiopulmonary bypass, including intestinal I/R. RESULTS: Succinate accumulated in lungs after intestinal I/R, and this was associated with an imbalance of succinate-producing and succinate-consuming bacteria in the gut, but not the lungs. Succinate accumulation was absent in germ-free mice and was reversed by gut microbiota depletion with antibiotics, indicating that the gut microbiota is a source of lung succinate. Moreover, succinate promoted alveolar macrophage polarisation, alveolar epithelial apoptosis and lung injury during intestinal I/R. Conversely, knockdown of Sucnr1 or blockage of SUCNR1 in vitro and in vivo reversed the effects of succinate by modulating the phosphoinositide 3-kinase-AKT/hypoxia-inducible factor-1α pathway. Plasma succinate levels significantly correlated with intestinal I/R-related lung injury after cardiopulmonary bypass. CONCLUSION: Gut microbiota-derived succinate exacerbates intestinal I/R-induced ALI through SUCNR1-dependent alveolar macrophage polarisation, identifying succinate as a novel target for gut-derived ALI in critically ill patients.
Subject(s)
Acute Lung Injury , Gastrointestinal Microbiome , Reperfusion Injury , Mice , Animals , Succinic Acid/metabolism , Phosphatidylinositol 3-Kinases , RNA, Ribosomal, 16S/genetics , Acute Lung Injury/complications , Reperfusion Injury/complications , Reperfusion Injury/metabolism , Reperfusion , Ischemia/complications , Mice, Inbred C57BLABSTRACT
Sepsis is associated with a high risk of death, and the crosstalk between gut microbiota and sepsis is gradually revealed. Indole 3-propionic acid (IPA) is a gut microbiota-derived metabolite that exerts immune regulation and organ protective effects. However, the role of IPA in sepsis is not clear. In this study, the role of IPA in sepsis-related survival, clinical scores, bacterial burden, and organ injury was assessed in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Aryl hydrocarbon receptor (AhR) highly specific inhibitor (CH223191) was used to observe the role of AhR in the protection of IPA against sepsis. The effects of IPA on bacterial phagocytosis by macrophages were investigated in vivo and vitro. The levels of IPA in feces were measured and analyzed in human sepsis patients and patient controls. First, we found that gut microbiota-derived IPA was associated with the survival of septic mice. Then, in animal model, IPA administration protected against sepsis-related mortality and alleviated sepsis-induced bacterial burden and organ injury, which was blunted by AhR inhibitor. Next, in vivo and vitro, IPA enhanced the macrophage phagocytosis through AhR. Depletion of macrophages reversed the protective effects of IPA on sepsis. Finally, on the day of ICU admission (day 0), septic patients had significantly lower IPA level in feces than patient controls. Also, septic patients with bacteremia had significantly lower IPA levels in feces compared with those with non-bacteremia. Furthermore, in septic patients, reduced IPA was associated with worse clinical outcomes, and IPA in feces had similar prediction ability of 28-day mortality with SOFA score, and increased the predictive ability of SOFA score. These findings indicate that gut microbiota-derived IPA can protect against sepsis through host control of infection by promoting macrophages phagocytosis and suggest that IPA may be a new strategy for sepsis treatment.
Subject(s)
Gastrointestinal Microbiome , Sepsis , Animals , Humans , Mice , Bacteria , Indoles/pharmacology , Macrophages , Mice, Inbred C57BL , Phagocytosis/physiology , Receptors, Aryl Hydrocarbon , Sepsis/microbiologyABSTRACT
BACKGROUND: Intestinal ischemia/reperfusion (I/R) injury has high morbidity and mortality rates. Gut microbiota is a potential key factor affecting intestinal I/R injury. Populations exhibit different sensitivities to intestinal I/R injury; however, whether this interpopulation difference is related to variation in gut microbiota is unclear. Here, to elucidate the interaction between the gut microbiome and intestinal I/R injury, we performed 16S DNA sequencing on the preoperative feces of C57BL/6 mice and fecal microbiota transplantation (FMT) experiments in germ-free mice. The transwell co-culture system of small intestinal organoids extracted from control mice and macrophages extracted from control mice or Toll-like receptor 2 (TLR2)-deficient mice or interleukin-10 (IL-10)-deficient mice were established separately to explore the potential mechanism of reducing intestinal I/R injury. RESULTS: Intestinal I/R-sensitive (Sen) and intestinal I/R-resistant (Res) mice were first defined according to different survival outcomes of mice suffering from intestinal I/R. Fecal microbiota composition and diversity prior to intestinal ischemia differed between Sen and Res mice. The relative abundance of Lactobacillus murinus (L. murinus) at the species level was drastically higher in Res than that in Sen mice. Clinically, the abundance of L. murinus in preoperative feces of patients undergoing cardiopulmonary bypass surgery was closely related to the degree of intestinal I/R injury after surgery. Treatment with L. murinus significantly prevented intestinal I/R-induced intestinal injury and improved mouse survival, which depended on macrophages involvement. Further, in vitro experiments indicated that promoting the release of IL-10 from macrophages through TLR2 may be a potential mechanism for L. murinus to reduce intestinal I/R injury. CONCLUSION: The gut microbiome is involved in the postoperative outcome of intestinal I/R. Lactobacillus murinus alleviates mice intestinal I/R injury through macrophages, and promoting the release of IL-10 from macrophages through TLR2 may be a potential mechanism for L. murinus to reduce intestinal I/R injury. This study revealed a novel mechanism of intestinal I/R injury and a new therapeutic strategy for clinical practice. Video Abstract.
Subject(s)
Reperfusion Injury , Toll-Like Receptor 2 , Animals , Humans , Interleukin-10 , Ischemia , Lactobacillus , Macrophages , Mice , Mice, Inbred C57BL , Reperfusion Injury/drug therapyABSTRACT
Intestinal ischemia/reperfusion (I/R) injury is a grave condition with high morbidity and mortality. We previously confirmed that intestinal I/R induces intestinal flora disorders and changes in metabolites, but the role of different metabolites in intestinal I/R injury is currently unclear. Based on targeted metabolic sequencing, pravastatin (PA) was determined to be a metabolite of the gut microbiota. Further, intestinal I/R model mice were established through superior mesenteric artery obstruction. In addition, a co-culture model of small intestinal organoids and type II innate lymphoid cells (ILC2s) was subjected to hypoxia/reoxygenation (H/R) to simulate an intestinal I/R model. Moreover, correlation analysis between the PA level in preoperative feces of patients undergoing cardiopulmonary bypass and the indices of postoperative intestinal I/R injury was carried out. IL-33-deficient mice, ILC2-deleted mice, and anti-IL-13 neutralizing antibodies were also used to explore the potential mechanism through which PA attenuates intestinal I/R injury. We demonstrated that PA levels in the preoperative stool of patients undergoing cardiopulmonary bypass were negatively correlated with the indices of postoperative intestinal I/R injury. Furthermore, PA alleviated intestinal I/R injury and improved the survival of mice. We further showed that PA promotes IL-13 release from ILC2s by activating IL-33/ST2 signaling to attenuate intestinal I/R injury. In addition, IL-13 promoted the self-renewal of intestinal stem cells by activating Notch1 and Wnt signals. Overall, results indicated that the gut microbial metabolite PA can attenuate intestinal I/R injury by promoting the release of IL-13 from ILC2s via IL-33/ST2 signaling, revealing a novel mechanism of and therapeutic strategy for intestinal I/R injury.