Cirrhosis-downregulated LSECtin can be retrieved by cytokines, shifts the TLR-induced LSECs secretome and correlates with the hepatic Th response.

BACKGROUND AND AIMS: We evaluated tolerogenic C-type lectin LSECtin loss in cirrhosis and its potential regulation by cytokines. METHODS: Liver tissue from patients with cirrhosis and healthy controls, immortalised and generated LSECtin-CRISPR immortalised LSECs, and murine primary LSECs from the CCl4 model were handled. RESULTS: LSECtin expression was reduced in liver tissue from cirrhotic patients, and it decreased from compensated to decompensated disease. Increased phosphorylation of MAPK, Akt and NFkB was observed upon LSECtin stimulation in LSEC murine cell line, showing a pattern of inflammatory and chemotactic cytokines either restrained (IL-10, CCL4) or unrestrained (TNF-α, IL-1ß, IL-6, CCL2). CD44 attenuated whereas LAG-3 increased all substrates phosphorylation in combination with TLR4 and TLR2 ligands except for NFkB. TNF-α, IL-1 ß, IL-6 and CCL2 were restrained by LSECtin crosslinking on TLRs studied. Conversely, IL-10 and CCL4 were upregulated, suggesting a LSECtin-TLRs synergistic effect. Also, LSECtin was significantly induced after IL-13 stimulation or combined with anti-inflammatory cytokines in cirrhotic and immortalised LSECs. Th17 and regulatory T cells were progressively increased in the hepatic tissue from compensated to decompensated patients. A significant inverse correlation was present between gene expression levels of CLEC4G/LSECtin and RORγT and FOXP3 in liver tissues. CONCLUSION: LSECtin restrains TLR proinflammatory secretome induced on LSECs by interfering immune response control, survival and MAPKs signalling pathways. The cytokine-dependent induction of LSECtin and the association between LSECtin loss and Th17 cell subset expansion in the liver, provides a solid background for exploring LSECtin retrieval as a mechanism to reprogram LSEC homeostatic function hampered during cirrhosis.

The 3Rs in Experimental Liver Disease.

Patients with cirrhosis present multiple physiological and immunological alterations that play a very important role in the development of clinically relevant secondary complications to the disease. Experimentation in animal models is essential to understand the pathogenesis of human diseases and, considering the high prevalence of liver disease worldwide, to understand the pathophysiology of disease progression and the molecular pathways involved, due to the complexity of the liver as an organ and its relationship with the rest of the organism. However, today there is a growing awareness about the sensitivity and suffering of animals, causing opposition to animal research among a minority in society and some scientists, but also about the attention to the welfare of laboratory animals since this has been built into regulations in most nations that conduct animal research. In 1959, Russell and Burch published the book "The Principles of Humane Experimental Technique", proposing that in those experiments where animals were necessary, everything possible should be done to try to replace them with non-sentient alternatives, to reduce to a minimum their number, and to refine experiments that are essential so that they caused the least amount of pain and distress. In this review, a comprehensive summary of the most widely used techniques to replace, reduce, and refine in experimental liver research is offered, to assess the advantages and weaknesses of available experimental liver disease models for researchers who are planning to perform animal studies in the near future.

Paneth Cells Regulate Lymphangiogenesis under Control of Microbial Signals during Experimental Portal Hypertension.

Intestinal microbiota can modulate portal hypertension through the regulation of the intestinal vasculature. We have recently demonstrated that bacterial antigens activate Paneth cells (PCs) to secrete products that regulate angiogenesis and portal hypertension. In the present work we hypothesized that Paneth cells regulate the development of lymphatic vessels under the control of intestinal microbiota during experimental portal hypertension. We used a mouse model of inducible PCs depletion (Math1Lox/LoxVilCreERT2) and performed partial portal vein ligation (PPVL) to induce portal hypertension. After 14 days, we performed mRNA sequencing and evaluated the expression of specific lymphangiogenic genes in small intestinal tissue. Intestinal and mesenteric lymphatic vessels proliferation was assessed by immunohistochemistry. Intestinal organoids with or without PCs were exposed to pathogen-associated molecular patterns, and conditioned media (CM) was used to stimulate human lymphatic endothelial cells (LECs). The lymphangiogenic activity of stimulated LECs was assessed by tube formation and wound healing assays. Secretome analysis of CM was performed using label-free proteomics quantification methods. Intestinal immune cell infiltration was evaluated by immunohistochemistry. We observed that the intestinal gene expression pattern was altered by the absence of PCs only in portal hypertensive mice. We found a decreased expression of specific lymphangiogenic genes in the absence of PCs during portal hypertension, resulting in a reduced proliferation of intestinal and mesenteric lymphatic vessels as compared to controls. In vitro analyses demonstrated that lymphatic tube formation and endothelial wound healing responses were reduced significantly in LECs treated with CM from organoids without PCs. Secretome analyses of CM revealed that PCs secrete proteins that are involved in lipid metabolism, cell growth and proliferation. Additionally, intestinal macrophages infiltrated the ileal mucosa and submucosa of mice with and without Paneth cells in response to portal hypertension. Our results suggest that intestinal microbiota signals stimulate Paneth cells to secrete factors that modulate the intestinal and mesenteric lymphatic vessels network during experimental portal hypertension.

Non-Alcoholic Fatty Liver Disease: Metabolic, Genetic, Epigenetic and Environmental Risk Factors.

Non-alcoholic fatty liver disease (NAFLD) is one of the most frequent causes of chronic liver disease in the Western world, probably due to the growing prevalence of obesity, metabolic diseases, and exposure to some environmental agents. In certain patients, simple hepatic steatosis can progress to non-alcoholic steatohepatitis (NASH), which can sometimes lead to liver cirrhosis and its complications including hepatocellular carcinoma. Understanding the mechanisms that cause the progression of NAFLD to NASH is crucial to be able to control the advancement of the disease. The main hypothesis considers that it is due to multiple factors that act together on genetically predisposed subjects to suffer from NAFLD including insulin resistance, nutritional factors, gut microbiota, and genetic and epigenetic factors. In this article, we will discuss the epidemiology of NAFLD, and we overview several topics that influence the development of the disease from simple steatosis to liver cirrhosis and its possible complications.

Liver Sinusoidal Endothelial Cells Contribute to Hepatic Antigen-Presenting Cell Function and Th17 Expansion in Cirrhosis.

Hepatic immune function is compromised during cirrhosis. This study investigated the immune features of liver sinusoidal endothelial cells (LSECs) in two experimental models of cirrhosis. Dendritic cells, hepatic macrophages, and LSECs were isolated from carbon tetrachloride and bile duct-ligated rats. Gene expression of innate receptors, bacterial internalization, co-stimulatory molecules induction, and CD4+ T cell activation and differentiation were evaluated. Induced bacterial peritonitis and norfloxacin protocols on cirrhotic rats were also carried out. LSECs demonstrated an active immunosurveillance profile, as shown by transcriptional modulation of different scavenger and cell-adhesion genes, and their contribution to bacterial internalization. LSECs significantly increased their expression of CD40 and CD80 and stimulated CD4+ T cell activation marker CD71 in both models. The pro-inflammatory Th17 subset was expanded in CCl4-derived LSECs co-cultures. In the bile duct ligation (BDL) model, CD4+ T cell differentiation only occurred under induced bacterial peritonitis conditions. Differentiated pro-inflammatory Th cells by LSECs in both experimental models were significantly reduced with norfloxacin treatment, whereas Foxp3 tolerogenic Th CD4+ cells were expanded. Conclusion: LSECs' participation in the innate-adaptive immune progression, their ability to stimulate pro-inflammatory CD4+ T cells expansion during liver damage, and their target role in norfloxacin-induced immunomodulation granted a specific competence to this cell population in cirrhosis.

How Valuable Is the RT-qPCR of Pituitary-Specific Transcription Factors for Identifying Pituitary Neuroendocrine Tumor Subtypes According to the New WHO 2017 Criteria?

The classification of pituitary neuroendocrine tumors (PitNETs) subtypes continues generating interest. In 2017, the World Health Organization (WHO) proposed considering the immunohistochemical (IHC) analysis of pituitary-specific transcription factors (TF) for their typification. The present study targeted the quantification of pituitary-specific TF (TPIT, PIT-1, SF-1, GATA2, ESR1) gene expression by RT-qPCR to overcome the shortcomings of IHC and to complement it. We analyzed 251 tumors from our collection of PitNETs and performed additional IHC studies in a subset of 56 samples to analyze the concordance between gene and protein expression of the TF. The molecular and IHC studies allowed us to significantly reduce the percentage of null cell tumors in our series, most of which were reclassified as gonadotroph tumors. The concordance between the molecular and the immunohistochemical studies was good for tumors coming from the corticotroph and Pit-1 lineages but worsened for the rest of the tumors. Indeed, the RT-qPCR helped to improve the typification of plurihormonal Pit-1 and unusual tumors. Overall, our results suggest that the RT-qPCR of pituitary-specific TF and hormone genes could help pathologists, endocrinologists, and neurosurgeons to improve the management of patients with pituitary tumors.

Members of the DEAL subfamily of the DUF1218 gene family are required for bilateral symmetry but not for dorsoventrality in Arabidopsis leaves.

Most plant leaves exhibit bilateral symmetry, which has been hypothesized as an inevitable consequence of the existence of the proximodistal and dorsoventral axes. No gene has been described that affects leaf bilateral symmetry but not dorsoventrality in Arabidopsis thaliana. We screened for viable insertional mutations that affect leaf morphology, and out of more than 700 mutants found only one, desigual1-1 (deal1-1), that exhibited bilateral symmetry breaking but no obvious defects in dorsoventrality. We found that deal1-1 is an allele of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC). Several overlapping regulatory pathways establish the interspersed lobes and indentations along the margin of Arabidopsis thaliana leaves. These pathways involve feedback loops of auxin, the PIN-FORMED1 (PIN1) auxin efflux carrier, and the CUP-SHAPED COTYLEDON2 (CUC2) transcriptional regulator. Early vcc (deal1) leaf primordia fail to acquire bilateral symmetry and instead form ectopic lobes and sinuses. The vcc leaves show aberrant recruitment of marginal cells expressing properly polarized PIN1, resulting in misplaced auxin maxima. Normal PIN1 polarization requires CUC2 expression and CUC2 genetically interacts with VCC; VCC also affects CUC2 expression. VCC has a domain of unknown function, DUF1218, and localizes to the endoplasmic reticulum membrane. VCC acts partially redundantly with its two closest paralogs, DEAL2 and DEAL3, in early leaf margin patterning and is required for bilateral symmetry, but its loss of function does not visibly affect dorsoventrality.