E3 Ubiquitin Ligase Midline 1 Regulates Endothelial Cell ICAM-1 Expression and Neutrophil Adhesion in Abdominal Sepsis

Septic lung damage is associated with endothelial cell and neutrophil activation. This study examines the role of the E3 ubiquitin ligase midline 1 (Mid1) in abdominal sepsis. Mid1 expression was increased in endothelial cells derived from post-capillary venules in septic mice and TNF-α challenge increased Mid1 levels in endothelial cells in vitro. The siRNA-mediated knockdown of Mid1 decreased TNF-α-induced upregulation of ICAM-1 and neutrophil adhesion to endothelial cells. Moreover, Mid1 silencing reduced leukocyte adhesion in post-capillary venules in septic lungs in vivo. The silencing of Mid1 not only decreased Mid1 expression but also attenuated expression of ICAM-1 in lungs from septic mice. Lastly, TNF-α stimulation decreased PP2Ac levels in endothelial cells in vitro, which was reversed in endothelial cells pretreated with siRNA directed against Mid1. Thus, our novel data show that Mid1 is an important regulator of ICAM-1 expression and neutrophil adhesion in vitro and septic lung injury in vivo. A possible target of Mid1 is PP2Ac in endothelial cells. Targeting the Mid1-PP2Ac axis may be a useful way to reduce pathological lung inflammation in abdominal sepsis.     

Targeting S100A9 Reduces Neutrophil Recruitment,Inflammation and Lung Damage in Abdominal Sepsis

S100A9, a pro-inflammatory alarmin, is up-regulated in inflamed tissues. However, the role of S100A9 in regulating neutrophil activation, inflammation and lung damage in sepsis is not known. Herein, we hypothesized that blocking S100A9 function may attenuate neutrophil recruitment in septic lung injury. Male C57BL/6 mice were pretreated with the S100A9 inhibitor ABR-238901 (10 mg/kg), prior to cercal ligation and puncture (CLP). Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested for analysis of neutrophil infiltration as well as edema and CXC chemokine production. Blood was collected for analysis of membrane-activated complex-1 (Mac-1) expression on neutrophils as well as CXC chemokines and IL-6 in plasma. Induction of CLP markedly increased plasma levels of S100A9. ABR-238901 decreased CLP-induced neutrophil infiltration and edema formation in the lung. In addition, inhibition of S100A9 decreased the CLP-induced up-regulation of Mac-1 on neutrophils. Administration of ABR-238901 also inhibited the CLP-induced increase of CXCL-1, CXCL-2 and IL-6 in plasma and lungs. Our results suggest that S100A9 promotes neutrophil activation and pulmonary accumulation in sepsis. Targeting S100A9 function decreased formation of CXC chemokines in circulation and lungs and attenuated sepsis-induced lung damage. These novel findings suggest that S100A9 plays an important pro-inflammatory role in sepsis and could be a useful target to protect against the excessive inflammation and lung damage associated with the disease.     

Benzoylpaeoniflorin Activates Anti-Inflammatory Mechanisms to Mitigate Sepsis in Cell-Culture and Mouse Sepsis Models

Xuebijing injection (XBJI) (comprising of five herbs) is a widely used traditional Chinese medicine for sepsis treatment. However, the bioactive components of XBJI and the mechanisms responsible for its sepsis-mitigating action have not been experimentally determined. One of the main bioactive compounds in XBJI—benzoylpaeoniflorin (BPF)—inhibits the expressions of key mediators of inflammation such as nuclear factor kappa B (NF-κB), cyclooxygenase-1 (COX-1), and COX-2. However, its effects on sepsis have not been determined yet. Therefore, here, we investigated the immunomodulatory effect of BPF on severely inflamed endothelial cells, THP-1 macrophages, peritoneal macrophages, and mice. Human umbilical vein endothelial cells (HUVECs) and THP-1-macrophages were activated using lipopolysaccharide (LPS) after pretreatment with BPF. Subsequently, changes in the expression profiles of pro-inflammatory molecules including inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were determined using quantitative real-time polymerase chain reaction (qPCR) and Western blot analysis. Furthermore, we monitored the phosphorylation of NF-kB and mitogen-activated protein kinases (MAPKs) to determine their activation levels. Using the LPS-induced mouse model of sepsis, we studied the effects of BPF on inflammatory cytokine production, pulmonary histopathology, and survival rates. Finally, we evaluated whether BPF protects against cecal ligation and puncture (CLP)-induced sepsis, as it closely mimics human sepsis. BPF pretreatment inhibited LPS-induced increase in mRNA and protein levels of iNOS, TNF-α, and IL-6 in HUVECs and THP-1-macrophages. It also suppressed LPS-mediated phosphorylation of p65, p38, JNK, and ERK. Mice with LPS-induced-sepsis who were treated with BPF had lower serum levels of IL-6, TNF-α, IL-1β, CXCL1, and CXCL2 than the control mice treated with BPF. Histopathology revealed that BPF treatment alleviated LPS-induced lung damage. In addition, in mice given a lethal dose of LPS, BPF treatment showed a dose-dependent improvement in survival rates. BPF treatment dose-dependently inhibited the LPS-induced IL-6, TNF-α, and CXCL1 production in peritoneal macrophages. BPF treatment also dose-dependently improved the survival rates in mice with CLP-induced sepsis. These results show that BPF alleviates LPS-stimulated septic conditions and protects mice from CLP-induced sepsis. Our research marks BPF as a potential drug in the treatment of sepsis and various inflammatory diseases.     

Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders

The heme molecule serves as an essential prosthetic group for oxygen transport and storage proteins, as well for cellular metabolic enzyme activities, including those involved in mitochondrial respiration, xenobiotic metabolism, and antioxidant responses. Dysfunction in both heme synthesis and degradation pathways can promote human disease. Heme is a pro-oxidant via iron catalysis that can induce cytotoxicity and injury to the vascular endothelium. Additionally, heme can modulate inflammatory and immune system functions. Thus, the synthesis, utilization and turnover of heme are by necessity tightly regulated. The microsomal heme oxygenase (HO) system degrades heme to carbon monoxide (CO), iron, and biliverdin-IXα, that latter which is converted to bilirubin-IXα by biliverdin reductase. Heme degradation by heme oxygenase-1 (HO-1) is linked to cytoprotection via heme removal, as well as by activity-dependent end-product generation (i.e., bile pigments and CO), and other potential mechanisms. Therapeutic strategies targeting the heme/HO-1 pathway, including therapeutic modulation of heme levels, elevation (or inhibition) of HO-1 protein and activity, and application of CO donor compounds or gas show potential in inflammatory conditions including sepsis and pulmonary diseases.     

Endothelial Dysfunction and Neutrophil Degranulation as Central Events in Sepsis Physiopathology

Sepsis is a major health problem worldwide. It is a time-dependent disease, with a high rate of morbidity and mortality. In this sense, an early diagnosis is essential to reduce these rates. The progressive increase of both the incidence and prevalence of sepsis has translated into a significant socioeconomic burden for health systems. Currently, it is the leading cause of noncoronary mortality worldwide and represents one of the most prevalent pathologies both in hospital emergency services and in intensive care units. In this article, we review the role of both endothelial dysfunction and neutrophil dysregulation in the physiopathology of this disease. The lack of a key symptom in sepsis makes it difficult to obtain a quick and accurate diagnosis of this condition. Thus, it is essential to have fast and reliable diagnostic tools. In this sense, the use of biomarkers can be a very important alternative when it comes to achieving these goals. Both new biomarkers and treatments related to endothelial dysfunction and neutrophil dysregulation deserve to be further investigated in order to open new venues for the diagnosis, treatment and prognosis of sepsis.     

Single-Cell RNA Sequencing Reveals Heterogeneity and Functional Diversity of Lymphatic Endothelial Cells

Lymphatic endothelial cells (LECs) line the lymphatic vasculature and play a central role in the immune response. LECs have abilities to regulate immune transport, to promote immune cell survival, and to cross present antigens to dendritic cells. Single-cell RNA sequencing (scRNA) technology has accelerated new discoveries in the field of lymphatic vascular biology. This review will summarize these new findings in regard to embryonic development, LEC heterogeneity with associated functional diversity, and interactions with other cells. Depending on the organ, location in the lymphatic vascular tree, and micro-environmental conditions, LECs feature unique properties and tasks. Furthermore, adjacent stromal cells need the support of LECs for fulfilling their tasks in the immune response, such as immune cell transport and antigen presentation. Although aberrant lymphatic vasculature has been observed in a number of chronic inflammatory diseases, the knowledge on LEC heterogeneity and functional diversity in these diseases is limited. Combining scRNA sequencing data with imaging and more in-depth functional experiments will advance our knowledge of LECs in health and disease. Building the case, the LEC could be put forward as a new therapeutic target in chronic inflammatory diseases, counterweighting the current immune-cell focused therapies.     

Differential Expression of Inflammasome-Related Genes in Induced Pluripotent Stem-Cell-Derived Retinal Pigment Epithelial Cells with or without History of Age-Related Macular Degeneration

Inflammation is a key underlying factor of age-related macular degeneration (AMD) and inflammasome activation has been linked to disease development. Induced pluripotent stem-cell-derived retinal pigment epithelial cells (iPSC-RPE) are an attractive novel model system that can help to further elucidate disease pathways of this complex disease. Here, we analyzed the effect of dysfunctional protein clearance on inflammation and inflammasome activation in iPSC-RPE cells generated from a patient suffering from age-related macular degeneration (AMD) and an age-matched control. We primed iPSC-RPE cells with IL-1α and then inhibited both proteasomal degradation and autophagic clearance using MG-132 and bafilomycin A1, respectively, causing inflammasome activation. Subsequently, we determined cell viability, analyzed the expression levels of inflammasome-related genes using a PCR array, and measured the levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and MCP-1 secreted into the medium. Cell treatments modified the expression of 48 inflammasome-related genes and increased the secretion of mature IL-1β, while reducing the levels of IL-6 and MCP-1. Interestingly, iPSC-RPE from an AMD donor secreted more IL-1β and expressed more Hsp90 prior to the inhibition of protein clearance, while MCP-1 and IL-6 were reduced at both protein and mRNA levels. Overall, our results suggest that cellular clearance mechanisms might already be dysfunctional, and the inflammasome activated, in cells with a disease origin.     

Analysis of Dormancy-Associated Transcriptional Networks Reveals a Shared Quiescence Signature in Lung and Colorectal Cancer

Quiescent cancer cells (QCCs) are a common feature of solid tumors, representing a major obstacle to the long-term success of cancer therapies. We isolated QCCs ex vivo from non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) xenografts with a label-retaining strategy and compared QCCs gene expression profiles to identify a shared “quiescence signature”. Principal Component Analysis (PCA) revealed a specific component neatly discriminating quiescent and replicative phenotypes in NSCLC and CRC. The discriminating component showed significant overlapping, with 688 genes in common including ZEB2, a master regulator of stem cell plasticity and epithelial-to-mesenchymal transition (EMT). Gene set enrichment analysis showed that QCCs of both NSCLC and CRC had an increased expression of factors related to stemness/self renewal, EMT, TGF-β, morphogenesis, cell adhesion and chemotaxis, whereas proliferating cells overexpressed Myc targets and factors involved in RNA metabolism. Eventually, we analyzed in depth by means of a complex network approach, both the ‘morphogenesis module’ and the subset of differentially expressed genes shared by NCSLC and CRC. This allowed us to recognize different gene regulation network wiring for quiescent and proliferating cells and to underpin few genes central for network integration that may represent new therapeutic vulnerabilities. Altogether, our results highlight common regulatory pathways in QCCs of lung and colorectal tumors that may be the target of future therapeutic interventions.     

Transcriptomic Analysis in Human 3D Skin Model Injected with Resorbable Hyaluronic Acid Fillers Reveals Foreign Body Response

Usage of injectable dermal fillers applied for aesthetic purposes has extensively increased over the years. As such, the number of related adverse reactions has increased, including patients showing severe complications such as product migration, topical swelling and inflammatory reactions of the skin. In order to understand the underlying molecular events of these adverse reactions we performed a genome-wide gene expression study on the multi-cell type human Phenion^® Full-Thickness Skin Model exposed to five experimental hyaluronic acid (HA) preparations with increasing cross-linking degree, four commercial fillers from Perfectha^®, and non-resorbable filler Bio-Alcamid^®. In addition, we evaluated whether cross-linking degree or particle size of the HA-based fillers could be associated with the occurrence of adverse effects. In all cases, exposure to different HA fillers resulted in a clearly elevated gene expression of cytokines and chemokines related to acute inflammation as part of the foreign body response. Furthermore, for one experimental filler genes of OXPHOS complexes I-V were significantly down-regulated (adjusted p-value < 0.05), resulting in mitochondrial dysfunction which can be linked to over-expression of pro-inflammatory cytokines TNFα and IL-1β and chemokine CCL2. Our hypothesis that cross-linking degree or particle size of the HA-based fillers is related to the biological responses induced by these fillers could only partially be confirmed for particle size. In conclusion, our innovative approach resulted in gene expression changes from a human 3D skin model exposed to dermal fillers that mechanistically substantiate aforementioned adverse reactions, and thereby adds to the weight of evidence that these fillers may induce inflammatory and fibrotic responses.     

Inhibitory Activities of Rare Ginsenoside Rg4 on Cecal Ligation and Puncture-Induced Sepsis

Sepsis is an uncontrolled response to inflammatory infection and is associated with high levels of mortality and morbidity. Rg4 is a rare ginsenoside mainly found in the leaves of Panax ginseng C. A. Meyer and the major protopanaxatriol-type ginsenoside of black ginseng. In this study, we determined whether Rg4 affects cecal ligation and puncture (CLP)-induced sepsis. Animals were separated into the following six groups: control group, CLP-operated group, CLP plus maslinic acid (MA), and CLP plus Rg4 (5, 10, or 15 mg/kg). Survival rate, body weight changes, inflammatory cytokines, and histological analyses were assessed. Human endothelial cells were activated with the high-mobility group box 1 (HMGB1) protein and Rg4. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used to assess inflammation and gene expression, respectively. After CLP surgery, the Rg4-administered group exhibited a higher survival rate and body weight compared with the untreated control group. Rg4 treatment reduced cytokine levels, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β, as well as nitric oxide (NO) levels and renal inflammation. After Rg4 treatment of HMGB1-activated cells, the expressions of toll-like receptor (TLR) 4 and TNF-α were decreased, and the activation of phosphoinositide 3-kinase (PI3K)/AKT signaling increased cell viability. In summary, Rg4 inhibited inflammation and exhibited a protective effect against CLP-induced sepsis, thereby reinforcing cell survival against septic responses.     

Proteomic Study of Differential Protein Expression in Mouse Lung Tissues after Aerosolized Ricin Poisoning

Ricin is one of the most poisonous natural toxins from plants and is classified as a Class B biological threat pathogen by the Centers for Disease Control and Prevention (CDC) of U.S.A. Ricin exposure can occur through oral or aerosol routes. Ricin poisoning has a rapid onset and a short incubation period. There is no effective treatment for ricin poisoning. In this study, an aerosolized ricin-exposed mouse model was developed and the pathology was investigated. The protein expression profile in the ricin-poisoned mouse lung tissue was analyzed using proteomic techniques to determine the proteins that were closely related to the toxicity of ricin. 2D gel electrophoresis, mass spectrometry and subsequent biological functional analysis revealed that six proteins including Apoa1 apolipoprotein, Ywhaz 14-3-3 protein, Prdx6 Uncharacterized Protein, Selenium-binding protein 1, HMGB1, and DPYL-2, were highly related to ricin poisoning.     

Differential Inflammatory Responses in Cultured Endothelial Cells Exposed to Two Conjugated Linoleic Acids (CLAs) under a Pro-Inflammatory Condition

Conjugated linoleic acid (CLA) isomers have been shown to possess anti-atherosclerotic properties, which may be related to the downregulation of inflammatory pathways in different cell types, including endothelial cells (ECs). However, whether different CLA isomers have different actions is not entirely clear, with inconsistent reports to date. Furthermore, in cell culture studies, CLAs have often been used at fairly high concentrations. Whether lower concentrations of CLAs are able to affect EC responses is not clear. The aim of this study was to evaluate the effects of two CLAs (cis-9, trans-11 (CLA9,11) and trans-10, cis-12 (CLA10,12)) on the inflammatory responses of ECs. ECs (EA.hy926 cells) were cultured under standard conditions and exposed to CLAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments with 1 and 10 μM of each CLA, but not after treatment with 50 μM of CLA10,12. CLAs were incorporated into ECs in a concentration-dependent manner. CLA10,12 increased the levels of ICAM-1, IL-6, and RANTES in the culture medium, while CLA9,11 had null effects. Both CLAs (1 μM) decreased the appearance of NFκB1 mRNA, but only CLA9,11 maintained this downregulation at 10 μM. CLA10,12 had no effect on THP-1 cell adhesion to ECs while significantly decreasing the percentage of ECs expressing ICAM-1 and also levels of ICAM-1 expression per cell when used at 10 µM. Although CLA9,11 did not have any effect on ICAM-1 cell surface expression, it reduced THP-1 cell adhesion to the EA.hy926 cell monolayer at both concentrations. In summary, CLA10,12 showed some pro-inflammatory effects, while CLA9,11 exhibited null or anti-inflammatory effects. The results suggest that each CLA has different effects in ECs under a pro-inflammatory condition, highlighting the need to evaluate the effects of CLA isomers independently.