Extracellular vesicles in sepsis plasma mediate neuronal inflammation in the brain through miRNAs and innate immune signaling.

BACKGROUND: Neuroinflammation reportedly plays a critical role in the pathogenesis of sepsis-associated encephalopathy (SAE). We previously reported that circulating plasma extracellular vesicles (EVs) from septic mice are proinflammatory. In the current study, we tested the role of sepsis plasma EVs in neuroinflammation. METHODS: To track EVs in cells and tissues, HEK293T cell-derived EVs were labeled with the fluorescent dye PKH26. Cecal ligation and puncture (CLP) was conducted to model polymicrobial sepsis in mice. Plasma EVs were isolated by ultracentrifugation and their role in promoting neuronal inflammation was tested following intracerebroventricular (ICV) injection. miRNA inhibitors (anti-miR-146a, -122, -34a, and -145a) were applied to determine the effects of EV cargo miRNAs in the brain. A cytokine array was performed to profile microglia-released protein mediators. TLR7- or MyD88-knockout (KO) mice were utilized to determine the underlying mechanism of EVs-mediated neuroinflammation. RESULTS: We observed the uptake of fluorescent PKH26-EVs inside the cell bodies of both microglia and neurons. Sepsis plasma EVs led to a dose-dependent cytokine release in cultured microglia, which was partially attenuated by miRNA inhibitors against the target miRNAs and in TLR7-KO cells. When administered via the ICV, sepsis plasma EVs resulted in a marked increase in the accumulation of innate immune cells, including monocyte and neutrophil and cytokine gene expression, in the brain. Although sepsis plasma EVs had no direct effect on cytokine production or neuronal injury in vitro, the conditioned media (CM) of microglia treated with sepsis plasma EVs induced neuronal cell death as evidenced by increased caspase-3 cleavage and Annexin-V staining. Cytokine arrays and bioinformatics analysis of the microglial CM revealed multiple cytokines/chemokines and other factors functionally linked to leukocyte chemotaxis and migration, TLR signaling, and neuronal death. Moreover, sepsis plasma EV-induced brain inflammation in vivo was significantly dependent on MyD88. CONCLUSIONS: Circulating plasma EVs in septic mice cause a microglial proinflammatory response in vitro and a brain innate immune response in vivo, some of which are in part mediated by TLR7 in vitro and MyD88 signaling in vivo. These findings highlight the importance of circulating EVs in brain inflammation during sepsis.

Naringenin Promotes Gastrointestinal Motility in Mice by Impacting the SCF/c-Kit Pathway and Gut Microbiota.

Naringenin (NRG) is widely found in citrus fruits and has anti-inflammatory, hypoglycemic, and immunomodulatory effects. Previous studies have shown that NRG promotes gastrointestinal motility in mice constipation models, but there are few systematic evaluations of its effects on normal animals. This study first clarified the promotive effects of NRG on gastric emptying and small intestine propulsion (p < 0.01). NRG can also regulate the release of gastrointestinal hormones, including enhancing gastrin (GAS) and motilin (MTL) (p < 0.01), while reducing vasoactive intestinal peptide (VIP) secretion (p < 0.01). Using NRG to stimulate the isolated stomach, duodenum, and colon showed similar promotive effects to those observed in vivo (p < 0.01). A Western blot analysis indicated that this effect may be mediated by increasing the expression of stem cell factor (SCF) and its receptor (c-Kit) in these three segments, thus regulating their downstream pathways. It is worth noting that NRG can also increase the proportion of beneficial bacteria (Planococcaceae, Bacteroides acidifaciens, Clostridia_UCG-014) in the intestine and reduce the quantity of harmful bacteria (Staphylococcus). These findings provide a new basis for the application of NRG.

Metabolic reprograming mediated by tumor cell-intrinsic type I IFN signaling is required for CD47-SIRPα blockade efficacy.

Type I interferons have been well recognized for their roles in various types of immune cells during tumor immunotherapy. However, their direct effects on tumor cells are less understood. Oxidative phosphorylation is typically latent in tumor cells. Whether oxidative phosphorylation can be targeted for immunotherapy remains unclear. Here, we find that tumor cell responsiveness to type I, but not type II interferons, is essential for CD47-SIRPα blockade immunotherapy in female mice. Mechanistically, type I interferons directly reprogram tumor cell metabolism by activating oxidative phosphorylation for ATP production in an ISG15-dependent manner. ATP extracellular release is also promoted by type I interferons due to enhanced secretory autophagy. Functionally, tumor cells with genetic deficiency in oxidative phosphorylation or autophagy are resistant to CD47-SIRPα blockade. ATP released upon CD47-SIRPα blockade is required for antitumor T cell response induction via P2X7 receptor-mediated dendritic cell activation. Based on this mechanism, combinations with inhibitors of ATP-degrading ectoenzymes, CD39 and CD73, are designed and show synergistic antitumor effects with CD47-SIRPα blockade. Together, these data reveal an important role of type I interferons on tumor cell metabolic reprograming for tumor immunotherapy and provide rational strategies harnessing this mechanism for enhanced efficacy of CD47-SIRPα blockade.

Recovery of subcutaneous orbital fascia under preserved skin below the original crease in the repair of excessively high double-eyelid folds.

BACKGROUND: High-eyelid fold is a common complication of upper eyelid surgery. This study proposed a novel technique for correcting high-eyelid fold in Asian patients with little eyelid skin and thick orbital fascia to improve cosmetic outcomes and patient satisfaction. METHODS: We retrospectively analysed the clinical data of 38 patients with high-eyelid fold repaired at the Plastic Surgery Hospital, Chinese Academy of Medical Sciences, from July 2017 to April 2022. All patients were treated using this method of preserving the high-eyelid fold and reconstructing the subcutaneous orbital fascia. Postoperative outcomes were reviewed and analysed using objective measures and subjective surgeon and patient evaluations. RESULTS: Thirty-eight patients (71 eyes) were included. Postoperative follow-up was for 6-25 months. At 6 months postoperatively, the mean anatomic crease height decreased from 10.45 mm to 6.22 mm, mean pretarsal show decreased from 3.20 mm to 1.61 mm, and mean margin reflex distance in 1 of the 31 patients with ptosis increased from 2.93 mm to 3.87 mm (P < 0.001). The compliance rate between the surgeon's assessment and patient satisfaction was 89.5%, with 31 cases considered good by both the surgeon and patient and one case considered poor by both (undercorrected ptosis), though a satisfactory result was achieved after reoperation. Neither recurrence of the original crease nor serious complications were reported. CONCLUSION: By preserving high-eyelid folds and reconstructing the subcutaneous orbital fascia, unnaturally high- and deep-eyelid folds were converted to lower, nondepressed folds.

IL-33 Induces Cellular and Exosomal miR-146a Expression as a Feedback Inhibitor of Mast Cell Function.

IL-33 is an inflammatory cytokine that promotes allergic disease by activating group 2 innate lymphoid cells, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. Homeostatic control of IL-33 signaling is poorly understood. Because the IL-33 receptor, ST2, acts via cascades used by the TLR family, similar feedback mechanisms may exist. MicroRNA (miR)-146a is induced by LPS-mediated TLR4 signaling and serves as a feedback inhibitor. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow-derived mast cells (BMMCs). BMMCs transfected with a miR-146a antagonist or derived from miR-146a knockout mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. In vivo, miR-146a expression in plasma exosomes was elevated after i.p. injection of IL-33 in wild-type but not mast cell-deficient KitW-sh/W-sh mice. Finally, KitW-sh/W-sh mice acutely reconstituted with miR-146a knockout BMMCs prior to IL-33 challenge had elevated plasma IL-6 levels compared with littermates receiving wild-type BMMCs. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

Endothelial SIRPα signaling controls VE-cadherin endocytosis for thymic homing of progenitor cells.

Thymic homing of hematopoietic progenitor cells (HPCs) is tightly regulated for proper T cell development. Previously we have identified a subset of specialized thymic portal endothelial cells (TPECs), which is important for thymic HPC homing. However, the underlying molecular mechanism still remains unknown. Here, we found that signal regulatory protein alpha (SIRPα) is preferentially expressed on TPECs. Disruption of CD47-SIRPα signaling in mice resulted in reduced number of thymic early T cell progenitors (ETPs), impaired thymic HPC homing, and altered early development of thymocytes. Mechanistically, Sirpa-deficient ECs and Cd47-deficient bone marrow progenitor cells or T lymphocytes demonstrated impaired transendothelial migration (TEM). Specifically, SIRPα intracellular ITIM motif-initiated downstream signaling in ECs was found to be required for TEM in an SHP2- and Src-dependent manner. Furthermore, CD47 signaling from migrating cells and SIRPα intracellular signaling were found to be required for VE-cadherin endocytosis in ECs. Thus, our study reveals a novel role of endothelial SIRPα signaling for thymic HPC homing for T cell development.

Thymic Egress Is Regulated by T Cell-Derived LTßR Signal and via Distinct Thymic Portal Endothelial Cells.

Thymic blood vessels at the perivascular space (PVS) are the critical site for both homing of hematopoietic progenitor cells (HPCs) and egress of mature thymocytes. It has been intriguing how different opposite migrations can happen in the same place. A subset of specialized thymic portal endothelial cells (TPECs) associated with PVS has been identified to function as the entry site for HPCs. However, the cellular basis and mechanism underlying egress of mature thymocytes has not been well defined. In this study, using various conventional and conditional gene-deficient mouse models, we first confirmed the role of endothelial lymphotoxin beta receptor (LTßR) for thymic egress and ruled out the role of LTßR from epithelial cells or dendritic cells. In addition, we found that T cell-derived ligands lymphotoxin (LT) and LIGHT are required for thymic egress, suggesting a crosstalk between T cells and endothelial cells (ECs) for thymic egress control. Furthermore, immunofluorescence staining analysis interestingly showed that TPECs are also the exit site for mature thymocytes. Single-cell transcriptomic analysis of thymic endothelial cells suggested that TPECs are heterogeneous and can be further divided into two subsets depending on BST-1 expression level. Importantly, BST-1hi population is associated with thymic egressing thymocytes while BST-1lo/- population is associated with HPC settling. Thus, we have defined a LT/LIGHT-LTßR signaling-mediated cellular crosstalk regulating thymic egress and uncovered distinct subsets of TPECs controlling thymic homing and egress, respectively.

Type 3 Innate Lymphoid Cells Direct Goblet Cell Differentiation via the LT-LTßR Pathway during Listeria Infection.

As a specialized subset of intestinal epithelial cells (IECs), goblet cells (GCs) play an important role during the antibacterial response via mucin production. However, the regulatory mechanisms involved in GC differentiation and function during infection, particularly the role of immune cell-IEC cross-talk, remain largely unknown. In this study, using Villin∆Ltbr conditional knockout mice, we demonstrate that LTßR, expressed on IECs, is required for GC hyperplasia and mucin 2 (MUC2) expression during Listeria infection for host defense but not homeostatic maintenance in the naive state. Analysis of single gene-deficient mice revealed that the ligand lymphotoxin (LT), but not LIGHT, and type 3 innate lymphoid cells (ILC3s), but not conventional T cells, are required for MUC2-dependent Listeria control. Conditional deficiency of LT in ILC3s further confirmed the importance of LT signals derived from ILC3s. Lack of ILC3-derived LT or IEC-derived LTßR resulted in the defective expression of genes related to GC differentiation but was not correlated with IEC proliferation and cell death, which were found to be normal by Ki-67 and Annexin V staining. In addition, the alternative NF-κB signaling pathway (involving RelB) in IECs was found to be required for the expression of GC differentiation-related genes and Muc2 and required for the anti-Listeria response. Therefore, our data together suggest a previously unrecognized ILC3-IEC interaction and LT-LTßR-RelB signaling axis governing GC differentiation and function during Listeria infection for host defense.

Epithelial LTßR signaling controls the population size of the progenitors of medullary thymic epithelial cells in neonatal mice.

The establishment of T cell central tolerance critically relies on the development and maintenance of the medullary thymic epithelial cells (mTECs). Disrupted signaling of lymphotoxin beta receptor (LTßR) results in dramatically reduced mTEC population. However, whether LTßR directly or indirectly control mTECs remains undetermined; how LTßR controls this process also remain unclear. In this study, by utilizing K14-Cre × Ltbrfl/fl conditional knockout (cKO) mice, we show that epithelial intrinsic LTßR was essential for the mTEC development postnatally. Mechanistically, LTßR did not directly impact the proliferation or survival of mTECs; the maturation of mTECs from MHC-IIlo to MHC-IIhi stage was also unaltered in the absence of LTßR; interestingly, the number of mTEC progenitors (Cld3,4hiSSEA-1+) was found significantly reduced in LTßR cKO mice at the neonatal stage, but not at E18.5. Consequently, epithelial deficiency of LTßR resulted in significant defect of thymic negative selection as demonstrated using OT-I and RIP-OVA transgenic mouse system. In summary, our study clarifies the epithelial intrinsic role of LTßR on mTEC development and function; more importantly, it reveals a previously unrecognized function of LTßR on the control of the size of mTEC progenitor population.

LTßR controls thymic portal endothelial cells for haematopoietic progenitor cell homing and T-cell regeneration.

Continuous thymic homing of haematopoietic progenitor cells (HPCs) via the blood is critical for normal T-cell development. However, the nature and the differentiation programme of specialized thymic endothelial cells (ECs) controlling this process remain poorly understood. Here using conditional gene-deficient mice, we find that lymphotoxin beta receptor (LTßR) directly controls thymic ECs to guide HPC homing. Interestingly, T-cell deficiency or conditional ablation of T-cell-engaged LTßR signalling results in a defect in thymic HPC homing, suggesting the feedback regulation of thymic progenitor homing by thymic products. Furthermore, we identify and characterize a special thymic portal EC population with features that guide HPC homing. LTßR is essential for the differentiation and homeostasis of these thymic portal ECs. Finally, we show that LTßR is required for T-cell regeneration on irradiation-induced thymic injury. Together, these results uncover a cellular and molecular pathway that governs thymic EC differentiation for HPC homing.