Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

Autologous humanized PDX modeling for immuno-oncology recapitulates features of the human tumor microenvironment.

BACKGROUND: Interactions between immune and tumor cells are critical to determining cancer progression and response. In addition, preclinical prediction of immune-related drug efficacy is limited by interspecies differences between human and mouse, as well as inter-person germline and somatic variation. To address these gaps, we developed an autologous system that models the tumor microenvironment (TME) from individual patients with solid tumors. METHOD: With patient-derived bone marrow hematopoietic stem and progenitor cells (HSPCs), we engrafted a patient's hematopoietic system in MISTRG6 mice, followed by transfer of patient-derived xenograft (PDX) tissue, providing a fully genetically matched model to recapitulate the individual's TME. We used this system to prospectively study tumor-immune interactions in patients with solid tumor. RESULTS: Autologous PDX mice generated innate and adaptive immune populations; these cells populated the TME; and tumors from autologously engrafted mice grew larger than tumors from non-engrafted littermate controls. Single-cell transcriptomics revealed a prominent vascular endothelial growth factor A (VEGFA) signature in TME myeloid cells, and inhibition of human VEGF-A abrogated enhanced growth. CONCLUSIONS: Humanization of the interleukin 6 locus in MISTRG6 mice enhances HSPC engraftment, making it feasible to model tumor-immune interactions in an autologous manner from a bedside bone marrow aspirate. The TME from these autologous tumors display hallmarks of the human TME including innate and adaptive immune activation and provide a platform for preclinical drug testing.

Nucleic DHX9 cooperates with STAT1 to transcribe interferon-stimulated genes.

RNA helicase DHX9 has been extensively characterized as a transcriptional regulator, which is consistent with its mostly nucleic localization. It is also involved in recognizing RNA viruses in the cytoplasm. However, there is no in vivo data to support the antiviral role of DHX9; meanwhile, as a nuclear protein, if and how nucleic DHX9 promotes antiviral immunity remains largely unknown. Here, we generated myeloid-specific and hepatocyte-specific DHX9 knockout mice and confirmed that DHX9 is crucial for host resistance to RNA virus infections in vivo. By additional knockout MAVS or STAT1 in DHX9-deficient mice, we demonstrated that nucleic DHX9 plays a positive role in regulating interferon-stimulated gene (ISG) expression downstream of type I interferon. Mechanistically, upon interferon stimulation, DHX9 is directly bound to STAT1 and recruits Pol II to the ISG promoter region to participate in STAT1-mediated transcription of ISGs. Collectively, these findings uncover an important role for nucleic DHX9 in antiviral immunity.

Human neutrophil development and functionality are enabled in a humanized mouse model.

Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system in vivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony-stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted in existing MISTRG mice. By targeting the G-CSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.

[Effect of early mechanical ventilation on the expression of inflammatory factors and prognosis in patients with severe traumatic brain injury].

OBJECTIVE: To observe the effect of early mechanical ventilation on the expression of inflammatory factors and prognosis of patients with severe traumatic brain injury (sTBI). METHODS: From January 2017 to December 2020, 138 patients with sTBI admitted to the department of the emergency of Xinhua Hospital Chongming Branch were enrolled. Although some patients were admitted to hospital without acute respiratory failure, their Glasgow coma score (GCS) were less than 8, they bad risk of hypoxia, so early mechanical ventilation was required. According to the patient's condition and the willingness of family members, patients were divided into mechanical ventilation group (tracheal intubation mechanical ventilation) and conventional oxygen inhalation group (nasal catheter or mask oxygen inhalation) in the end. The arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2), oxygenation index (PaO2/FiO2), tumor necrosis factor-α (TNF-α), and interleukin (IL-6, IL-10) levels at admission, preoperation and 72 hours postoperation, as well as GCS before operation and 1 week after operation, the duration and number of patients successfully evacuated from the ventilator within 1 week after surgery were observed and analyzed. RESULTS: A total of 138 sTBI patients were enrolled in the study, including 72 cases in the mechanical ventilation group and 66 cases in the routine oxygen inhalation group. In the two groups, PaO2, PaO2/FiO2 and IL-10 were higher, and PaCO2, TNF-α and IL-6 were lower at 72 hours post operation than that before operation. Moreover, the changes in the mechanical ventilation group were more significant than those in the conventional oxygen inhalation group [PaO2 (1 mmHg = 0.133 kPa): 94.6±7.7 vs. 92.5±6.8, PaO2/FiO2 (mmHg): 351±94 vs. 319±89, IL-10 (ng/L): 8.2±2.7 vs. 7.4±1.8, PaCO2 (mmHg): 35.6±1.8 vs. 37.5±2.7, TNF-α(ng/L): 71.5±6.3 vs. 96.8±15.5, IL-6 (ng/L): 10.8±3.9 vs. 14.4±6.5, all P < 0.05]. There were 17 patients with severe respiratory insufficiency or failure in the conventional oxygen inhalation group before operation. Compared with the conventional oxygen inhalation group, the GCS score (11.7±3.1 vs. 9.1±4.6) and the proportion of successful weaning [62.5% (45/72) vs. 44.0% (29/66)] were significantly higher, and the duration of successful weaning (hours: 63.5±28.6 vs. 88.1±33.9) was significantly shorter in the mechanical ventilation group 1 week after operation. CONCLUSIONS: Early mechanical ventilation in sTBI patients can significantly improve oxygen supply, inhibit the release of pro-inflammatory factors, reduce secondary brain damage, and effectively improve the prognosis.

Anti-SIRPα antibody immunotherapy enhances neutrophil and macrophage antitumor activity.

Cancer immunotherapy has emerged as a promising therapeutic intervention. However, complete and durable responses are only seen in a fraction of patients who have cancer. A key factor that limits therapeutic success is the infiltration of tumors by cells of the myeloid lineage. The inhibitory receptor signal regulatory protein-α (SIRPα) is a myeloid-specific immune checkpoint that engages the "don't eat me" signal CD47 expressed on tumors and normal tissues. We therefore developed the monoclonal antibody KWAR23, which binds human SIRPα with high affinity and disrupts its binding to CD47. Administered by itself, KWAR23 is inert, but given in combination with tumor-opsonizing monoclonal antibodies, KWAR23 greatly augments myeloid cell-dependent killing of a collection of hematopoietic and nonhematopoietic human tumor-derived cell lines. Following KWAR23 antibody treatment in a human SIRPA knockin mouse model, both neutrophils and macrophages infiltrate a human Burkitt's lymphoma xenograft and inhibit tumor growth, generating complete responses in the majority of treated animals. We further demonstrate that a bispecific anti-CD70/SIRPα antibody outperforms individually delivered antibodies in specific types of cancers. These studies demonstrate that SIRPα blockade induces potent antitumor activity by targeting multiple myeloid cell subsets that frequently infiltrate tumors. Thus, KWAR23 represents a promising candidate for combination therapy.

Nlrp9b inflammasome restricts rotavirus infection in intestinal epithelial cells.

Rotavirus, a leading cause of severe gastroenteritis and diarrhoea in young children, accounts for around 215,000 deaths annually worldwide. Rotavirus specifically infects the intestinal epithelial cells in the host small intestine and has evolved strategies to antagonize interferon and NF-κB signalling, raising the question as to whether other host factors participate in antiviral responses in intestinal mucosa. The mechanism by which enteric viruses are sensed and restricted in vivo, especially by NOD-like receptor (NLR) inflammasomes, is largely unknown. Here we uncover and mechanistically characterize the NLR Nlrp9b that is specifically expressed in intestinal epithelial cells and restricts rotavirus infection. Our data show that, via RNA helicase Dhx9, Nlrp9b recognizes short double-stranded RNA stretches and forms inflammasome complexes with the adaptor proteins Asc and caspase-1 to promote the maturation of interleukin (Il)-18 and gasdermin D (Gsdmd)-induced pyroptosis. Conditional depletion of Nlrp9b or other inflammasome components in the intestine in vivo resulted in enhanced susceptibility of mice to rotavirus replication. Our study highlights an important innate immune signalling pathway that functions in intestinal epithelial cells and may present useful targets in the modulation of host defences against viral pathogens.

Expression of HMGB1 in septic serum induces vascular endothelial hyperpermeability.

The aim of the present study was to investigate the effects of high mobility group protein B1 (HMGB1), which is expressed in the serum of patients with sepsis, on vascular endothelial permeability. Sera from patients with sepsis were used to treat endothelial cells (ECs), and the effect on endothelial permeability was evaluated using immunofluorescence. The morphologies of endothelial cytoskeletal actin and vascular endothelial (VE)­cadherin were assessed using laser scanning confocal microscopy. The protein expression levels of HMGB1, B­cell lymphoma 2 (BCL­2) and BCL­2­associated X protein (BAX) were detected using western blotting. EC apoptosis was measured using flow cytometry. The results demonstrated that HMGB1 was significantly expressed in the serum 24 h following the onset of sepsis, and the expression levels peaked at 48 h, which were sustained until 96 h post­onset. Compared with the control group, treatment of the ECs with 20% septic serum in vitro significantly increased endothelial monolayer permeability (P<0.01), markedly induced transcellular filamentous (F)­actin rearrangement with stress fiber formation, and resulted in the localization of VE­cadherin fragmentations at the cell borders with increased gaps between ECs. Furthermore, flow cytometry showed that the apoptotic rate of ECs was significantly increased following treatment with septic serum. In addition, the expression levels of BAX were significantly increased, whereas the expression levels of BCL­2 were significantly decreased. Pretreatment with an HMGBI inhibitor (ethyl pyruvate; 5 µM) 24 h prior to treatment with the septic serum attenuated the effects of septic serum treatment. Together, these findings suggested that treatment of ECs with sera from patients with sepsis may induce the loss of vascular endothelial monolayer integrity, elicit the formation of endothelial F­actin stress fibers and initiate VE­cadherin redistribution, which may be attributed to high levels of HMGB1 in the serum. This mechanism also appears to involve changes in the activation of BAX and BCL­2, resulting in EC apoptosis.

Nlrp6 regulates intestinal antiviral innate immunity.

The nucleotide-binding oligomerization domain-like receptor (Nlrp) 6 maintains gut microbiota homeostasis and regulates antibacterial immunity. We now report a role for Nlrp6 in the control of enteric virus infection. Nlrp6(-/-) and control mice systemically challenged with encephalomyocarditis virus had similar mortality; however, the gastrointestinal tract of Nlrp6(-/-) mice exhibited increased viral loads. Nlrp6(-/-) mice orally infected with encephalomyocarditis virus had increased mortality and viremia compared with controls. Similar results were observed with murine norovirus 1. Nlrp6 bound viral RNA via the RNA helicase Dhx15 and interacted with mitochondrial antiviral signaling protein to induce type I/III interferons (IFNs) and IFN-stimulated genes (ISGs). These data demonstrate that Nlrp6 functions with Dhx15 as a viral RNA sensor to induce ISGs, and this effect is especially important in the intestinal tract.

MiR-125a targets effector programs to stabilize Treg-mediated immune homeostasis.

Although different autoimmune diseases show discrete clinical features, there are common molecular pathways intimately involved. Here we show that miR-125a is downregulated in peripheral CD4(+) T cells of human autoimmune diseases including systemic lupus erythematosus and Crohn's disease, and relevant autoimmune mouse models. miR-125a stabilizes both the commitment and immunoregulatory capacity of Treg cells. In miR-125a-deficient mice, the balance appears to shift from immune suppression to inflammation, and results in more severe pathogenesis of colitis and experimental autoimmune encephalomyelitis (EAE). The genome-wide target analysis reveals that miR-125a suppresses several effector T-cell factors including Stat3, Ifng and Il13. Using a chemically synthesized miR-125a analogue, we show potential to re-programme the immune homeostasis in EAE models. These findings point to miR-125a as a critical factor that controls autoimmune diseases by stabilizing Treg-mediated immune homeostasis.

Proinflammatory effects and molecular mechanisms of interleukin-17 in intestinal epithelial cell line HT-29.

AIM: To evaluate the proinflammatory effects and molecular mechanisms of interleukin (IL)-17 in intestinal epithelial cell line HT-29. METHODS: HT-29 cells were cultured with IL-17, tumor necrosis factor (TNF)-α, or the combination of both IL-17 and TNF-α. Real-time PCR and Western blot were used to measure the gene expression levels of neutrophil chemokines CXCL1, CXCL2, CXCL5, CXCL6, IL-8 and TH-17 cell chemokine CCL20, the phosphorylation levels of p38 and TNF-α, and the expression level of IL-8, after using the p38 inhibitor in HT-29 cells. The stable Act1 knockdown HT-29 cell line was established to further test the phosphorylation changes of p38, after using IL-17 and TNF-α. RESULTS: After HT-29 cells were cultured with IL-17 and TNF-α, the expression levels of neutrophil chemokines (CXCL1, CXCL2, CXCL5, CXCL6, IL-8) and Th17 chemokine (CCL20) significantly improved (24.96 ± 2.53, 28.47 ± 2.87, 38.08 ± 2.72, 33.47 ± 2.41, 31.7 ± 2.38, 44.37 ± 2.73, respectively), and the differences were all statistically significant (P < 0.01). Western blot results showed that IL-17 obviously enhanced the phosphorylation level of p38, which was induced by TNF-α. Compared with the control group, the expression level of IL-8 significantly declined (9.47 ± 1.36 vs 3.06 ± 0.67, P < 0.01) when TH-29 cells were cultured with IL-17 and TNF-α. p38 inhibition assay showed that the p38 pathway played an essential role in the inflammatory response induced by IL-17. p38 phosphorylation levels could not be changed after using IL-17 and TNF-α in the stable Act1 knockdown HT-29 cell line. CONCLUSION: IL-17 significantly promoted the gene expression levels of TNF-α-induced neutrophil chemokines and Th17 cell chemokine. It is obvious that IL-17 and TNF-α have synergistic effects on p38.

Effect of serum from patients with severe acute pancreatitis on vascular endothelial permeability.

OBJECTIVE: This study aimed to investigate the effect of serum taken from patients with severe acute pancreatitis (SAP) on vascular endothelial permeability. METHODS: The monolayer permeability of endothelial cells (ECs) was assessed. Morphological changes in ECs, induced by serum from patients with SAP were assessed. Expressions of RhoA, myosin light chain (MLC) phosphorylation, and VE-cadherin protein were detected by Western blot. RESULTS: Compared with the control group, 20% SAP serum significantly increased endothelial monolayer permeability (P < 0.01), markedly induced transcellular F-actin redistribution with stress fiber formation and VE-cadherin derangement with fragmentations located at the cell borders, and increased gaps between ECs. Furthermore, Western blotting showed that SAP serum induced rapid activation of Rho protein, and markedly increased the level of phosphorylated MLC. However, pretreatment with Y-27632 (an inhibitor for Rho kinase) significantly inhibited endothelial hyperpermeability and the morphological changes of F-actin rearrangement and VE-cadherin redistribution. This was associated with a down-regulation of Rho protein expression and a reduction in the level of MLC phosphorylation. CONCLUSIONS: The SAP serum induces the loss of vascular endothelial monolayer integrity, with endothelial F-actin stress fiber formation and VE-cadherin redistribution. One of the mechanisms for this process involves the activation of the Rho/Rho kinase signaling pathway.

Study of Astragalus mongholicus polysaccharides on endothelial cells permeability induced by HMGB1.

Astragalus mongholicus polysaccharide (APS) shows various biological activities. Here, we explored the effect of APS on high mobility group protein 1 (HMGB1) -induced endothelial cell permeability. The results indicated APS pretreatment effectively inhibited HMGB1-induced increased permeability in endothelial cells (ECs). Signal transduction studies showed APS inhibited not only the activation of small guanylate Rho and its downstream effector Rho kinase (ROCK), but also the subsequent phosphorylation of myosin light chain (MLC) in ECs. In conclusion, our investigations suggested that APS inhibited HMGB1-induced increased permeability in ECs, regulated by Rho/ROCK signal pathways.

[A study on increased permeability and morphological changes in actin cytoskeleton and tight junction of vascular endothelial cells induced by tumor necrosis factor-alpha].

OBJECTIVE: To study the effect of human tumor necrosis factor-alpha (TNF-alpha) on permeability of human vascular endothelial cell (EA.hy926) monolayer and its mechanism. METHODS: 5, 10, 20 microg/L TNF-alpha was respectively added to the cultured endothelial cell monolayer for 24 hours, or 10 microg/L TNF-alpha for 6, 12, 24 hours. Human vascular endothelial cell (EA.hy926) monolayer permeability was measured by detecting fluorescence intensity of fluorescein isothiocyanate (FITC) labeled dextran. Immunofluorescence and laser confocal microscopy were used to assess vascular endothelial actin cytoskeleton (F-actin) and tight junction protein (ZO-1) distribution. Western blotting was used to assess ZO-1 expression. RESULTS: Compared with control group, TNF-alpha significantly increased endothelial permeability and induced F-actin redistribution and stress fiber formation with ZO-1 derangement. Gaps increased obviously between endothelial cells. Furthermore, Western blotting showed that TNF-alpha reduced ZO-1 expression in a dose- and time-dependent manner. CONCLUSION: TNF-alpha increased endothelial cell permeability by damaging integrity of endothelial barrier function.

Surgical experience of refined 3-cuff technique for orthotopic small-bowel transplantation in rat: a report of 270 cases.

BACKGROUND: Establishment of an instant, reproducible, and reliable rat model of a refined 3-cuff technique for performing orthotopic intestinal transplantation is reported, and the surgical skills required to perform modified surgical procedure are discussed. METHODS: A retrospective analysis was used to study 270 rat cases subject to orthotopic intestinal transplantation (OIT) performed in our transplantation center from March 2006 to March 2008. After establishing the portal vein cuff method, a conventional hand-sewn anastomosis method combination, with porto-to-portal re-establishment by cuffed anastomosis technique, was used in group 1 (n = 140), and the modified 3-cuff anastomosis method was applied in group 2 (n = 130). Statistical comparison was made between the 2 groups. RESULTS: In group 1, 97 of 140 (69.3%) recipients survived >7 days, and 69 (49.3%) survived >30 days, whereas in group 2, respective survival was 110 of 130 (84.6%) and 86 of 130 (66.2%). Average cold ischemic times in the 2 groups were 48.5 +/- 5.1 minutes and 31 +/- 3.0 minutes, respectively. There was a significant difference between the 2 groups (P <.05). In most cases, the average volume of bleeding during recipient surgery was <1 mL using the simplified 3-cuff anastomosis technique. There was shorter graft revascularization time with the new model of sutureless microanastomosis using cuff apparatus for OIT in rats compared with the control group. The method adopted in group 2 was much easier, more stable, and more feasible than that in group 1. Sixty-three rats died in 7 days, and autopsy verified the causative factors leading to death, which are summarized in the text. The results obtained were acceptable and satisfactory. Overall, there was a comparative lower incidence of complications associated with the procedure used in group 2. CONCLUSIONS: The modified 3-cuff anastomosis technique for rat OIT models has several obvious advantages, which can be summarized as follows: vascular anastomosis is stable and simplified, and blood loss is significantly decreased; natural anatomic physiologic portal graft drainage is maintained; and intraoperative mortality and postsurgical morbidity are minimized. Furthermore, technical refinement of rat OIT models established by our research team can be carried out without a microscope and can be easily implemented in the laboratory by 1 trainee with acceptable success after a short period of training. We regard it as one of the best available orthotopic small-bowel transplantation methods in rat.