Liver Lymphocytes Research Articles

The transcription factor T-bet promotes the pathogenesis of nonalcoholic fatty liver disease by upregulating intrahepatic inflammation

ObjectiveTo investigate the effect of the transcription factor T-bet on the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and the regulation of the intrahepatic immune microenvironment. MethodsWild-type and T-bet knockout NASH mouse models were constructed. The effect of T-bet knockout on the pathogenesis of NAFLD was observed by histochemical staining. The expression of T-bet in immune cells in the liver and the effect of T-bet knockout on the proportion and function of immune cell subsets in the liver were determined by flow analysis. ResultsFlow cytometry results indicated that T-bet expression was increased in immune cells, especially NKT cells, in the livers of NAFLD mice. Knocking out the transcription factor T-bet reduced intrahepatic inflammation, reduced lipid accumulation, and ameliorated the pathogenesis of NAFLD. Based on the analysis of immune cell subsets, knocking out the transcription factor T-bet decreased the proportion, survival, and degree of activation of NK, NKT, and CD8 T cells in NAFLD liver; additionally, it decreased the secretion of IFN-γ by T cells and NKT cells but had no effect on the proportion of Th17 cells and Treg cells. Knocking out the transcription factor T-bet also reduced the proportion of proinflammatory myeloid-derived macrophages (MoMFs) in NAFLD liver, mainly the proportion of proinflammatory Ly6Chigh MoMFs. Furthermore, knocking out the transcription factor T-bet had no significant effect on the secretion of TNF-α from MoMFs but significantly reduced the expression of MHC class II molecules. Further analysis showed that the transcription factor T-bet may directly affect the expression of MHC class II molecules H2-AB1 and H2-Dmb1 through transcriptional regulation. ConclusionsKnocking out the transcription factor T-bet reduced the proinflammatory effect of innate immune cells (MoMFs, NK cells, and NKT cells) and T lymphocytes in NAFLD liver, thereby reducing intrahepatic inflammation and delaying the pathogenesis of NAFLD.

DNAM-1 Immunoreceptor Protects Mice from Concanavalin A-Induced Acute Liver Injury by Reducing Neutrophil Infiltration.

DNAX accessory molecule-1 (DNAM-1; CD226) is an activating immunoreceptor on T cells and NK cells. The interaction of DNAM-1 with its ligand CD155 expressed on hematopoietic and nonhematopoietic cells plays an important role in innate and adaptive immune responses. In this study, we investigated the role of the DNAM-1-CD155 axis in the pathogenesis of T cell-mediated Con A-induced acute liver injury. Unexpectedly, DNAM-1-deficient (Cd226-/-) mice exhibited more severe acute liver injury and higher concentrations of IL-6 and TNF-α than did wild-type (WT) mice after Con A injection. We found that a larger number of neutrophils infiltrated into the liver of Cd226-/- mice compared with WT mice after Con A injection. Depletion of neutrophils ameliorated liver injury and decreased IL-6 and TNF-α in Cd226-/- mice after Con A injection, suggesting that neutrophils exacerbate the liver injury in Cd226-/- mice. Hepatocytes produced more significant amounts of CXCL1, a chemoattractant for neutrophils, in Cd226-/- mice than in WT mice after Con A injection. In the coculture of hepatocytes with liver lymphocytes, either DNAM-1 deficiency in liver lymphocytes or CD155 deficiency in hepatocytes promoted CXCL1 production by hepatocytes. These results suggest that the interaction of DNAM-1 with CD155 inhibits CXCL1 production by hepatocytes, leading to ameliorating acute liver injury.

Alleviation of Hepatic Steatosis by Alpha-Defensin Is Associated with Enhanced Lipolysis.

Background and Objectives: The neutrophilic peptide, alpha-defensin, is considered an evolving risk factor intimately linked with lipid mobilization. It was previously linked to augmented liver fibrosis. Here, we assess a potential association between alpha-defensin and fatty liver. Materials and Methods: A cohort of transgenic C57BL/6JDef+/+ male mice that overexpress the human neutrophil-derived alpha-defensin in their polymorphonuclear neutrophils (PMNs) were assessed for liver steatosis and fibrosis development. Wild type (C57BL/6JDef.Wt) and transgenic (C57BL/6JDef+/+) mice were maintained on a standard rodent chow diet for 8.5 months. At the termination of the experiment, systemic metabolic indices and hepatic immunological cell profiling were assessed. Results: The Def+/+ transgenic mice exhibited lower body and liver weights, lower serum fasting glucose and cholesterol, and significantly lower liver fat content. These results were associated with impaired liver lymphocytes count and function (lower CD8, NK cells, and killing marker CD107a). The metabolic cage demonstrated dominant fat utilization with a comparable food intake in the Def+/+ mice. Conclusions: Chronic physiological expression of alpha-defensin induces favorable blood metabolic profile, increased systemic lipolysis, and decreased hepatic fat accumulation. Further studies are needed to characterize the defensin net liver effect.

Abstract 5465: Immune cell dynamics of patients and mice with hepatocellular carcinoma treated with anti-PD-L1 plus anti-CTLA-4 combination therapy

Abstract Background: Hepatocellular carcinoma (HCC) is one of the major causes for cancer-related death. Single agent immunotherapy or combined immunotherapy have become the standard of care in advanced HCC. Recently, the combination of durvalumab (dur) plus a single dose of 300 mg tremelimumab (trem) (STRIDE regimen) have received FDA approval based on results from the HIMALAYA trial. However, immune responses have not been studied in detail. We tested the combination of 4 × 75 mg trem plus dur and locoregional therapies in HCC at and conducted correlative studies. Methods: We enrolled 28 patients with biopsy proven advanced HCC in an open-labeled phase 2 trial using dur and trem combined with ablative therapies (NCT02821754). Patients received four doses of 75mg trem every 4 weeks and 1500mg dur every 4 weeks until progression. Patients underwent an interventional radiologic procedure (RFA/TACE) on day 36. Peripheral blood mononuclear cells (PBMC) and serum from patients enrolled were collected at baseline and after 1 cycle (day 28). PBMC was analyzed by multicolor spectral cytometry using a 17 antibody pan immune cell panel and a 25 antibody T cell panel. Serum samples from patients were collected at baseline and on day 28. Serum samples were analyzed with a 34-plex cytokine/chemokine array. Mice with orthotopically injected HCC (RIL-175 and Hep55.1C) were treated with anti-PD-L1 plus anti-CTLA4 and followed for tumor responses. Hepatic lymphocytes, tumor infiltrating lymphocytes (TILs), and splenocytes were analyzed by spectral cytometry from mice treated with immune checkpoint inhibitors. Results: 28 patients with advanced HCC were enrolled in this study. The best clinical response for treatment was the following, partial response (PR, n=5), stable disease (SD, n=12), progressive disease (PD, n=8), and not evaluable (n=3). Median PFS and OS were 4.5 and 20.8 months respectively. Patient PBMC analysis demonstrated an increase of regulatory T cells 1.65 times from baseline after 28 days in all patients. The frequency of Tbet+CD4+ T cells showed a non-significant increase in patients demonstrating a PR. Serum cytokine analysis revealed the levels of IL-18, IL-10, CCL-2, and VEGF-a increased in responders after treatment. in contrast, an increase of IL-6 and TNF-alpha after treatment was found only in patients with PD. In animal experiments, the size of liver tumors treated with anti-PDL1 and anti-CTLA4 were significantly smaller than that with isotype controls. The lymphocytes in the adjacent livers and tumor-infiltrating lymphocytes (TILs) were analyzed. PD1+CD8+ T cells and Tbet+CD4+ T cells in the liver and TILs increased with combination treatment. Conclusion: Anti-PD-L1 and anti-CTLA4 combination therapy have an anti-tumor effect on HCC. We observed an expansion of PD1+ CD8+ T cells and Tbet+ CD4+ T cells. Citation Format: Yuta Myojin, Benjamin Ruf, Mohamed-Reda Benmebarek, Kylynda Bauer, Rajiv Trehan, Kelley Coffman, Chi Ma, Cecilia B. Monge, Changqing Xie, Tim Greten. Immune cell dynamics of patients and mice with hepatocellular carcinoma treated with anti-PD-L1 plus anti-CTLA-4 combination therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5465.

Single-cell transcriptome analysis uncovers underlying mechanisms of acute liver injury induced by tripterygium glycosides tablet in mice

Tripterygium glycosides tablet (TGT), the classical commercial drug of Tripterygium wilfordii Hook. F. has been effectively used in the treatment of rheumatoid arthritis, nephrotic syndrome, leprosy, Behcet's syndrome, leprosy reaction and autoimmune hepatitis. However, due to its narrow and limited treatment window, TGT-induced organ toxicity (among which liver injury accounts for about 40% of clinical reports) has gained increasing attention. The present study aimed to clarify the cellular and molecular events underlying TGT-induced acute liver injury using single-cell RNA sequencing (scRNA-seq) technology. The TGT-induced acute liver injury mouse model was constructed through short-term TGT exposure and further verified by hematoxylin-eosin staining and liver function-related serum indicators, including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and total bilirubin. Using the mouse model, we identified 15 specific subtypes of cells in the liver tissue, including endothelial cells, hepatocytes, cholangiocytes, and hepatic stellate cells. Further analysis indicated that TGT caused a significant inflammatory response in liver endothelial cells at different spatial locations; led to marked inflammatory response, apoptosis and fatty acid metabolism dysfunction in hepatocytes; activated hepatic stellate cells; brought about the activation, inflammation, and phagocytosis of liver capsular macrophages cells; resulted in immune dysfunction of liver lymphocytes; disturbed the intercellular crosstalk in liver microenvironment by regulating various signaling pathways. Thus, these findings elaborate the mechanism underlying TGT-induced acute liver injury, provide new insights into the safe and rational applications in the clinic, and complement the identification of new biomarkers and therapeutic targets for liver protection.

Single-cell RNA-seq Revealed that Altered Tumor Infiltrating Lymphocytes in Cirrhotic Liver Indicate Development of Hepatocellular Carcinoma.

Cirrhosis is the precursor lesion for most hepatocellular carcinoma (HCC) cases. However, no biomarker effectively predicted HCC initiation before diagnosis by imaging. We aimed to investigate the hallmarks of immune microenvironments in healthy, cirrhotic livers and HCC tumor tissues and to identify immune biomarkers of cirrhosis-HCC transition. Expression matrices of single-cell RNA sequencing studies were downloaded and integrated with Seurat package vignettes. Clustering was performed to analyze the immune cell compositions of different sample types. The cirrhotic liver and HCC tumors had distinct immune microenvironments, but the immune landscape of cirrhotic livers was not markedly modified compared with healthy livers. Two subsets of B cells and three subsets of T cells were identified in the samples. Among the T cells, naïve T cells were more prominent in the cirrhotic and healthy liver samples than in the HCC samples. In contrast, the neutrophil count was lower in cirrhotic livers. Two macrophage clusters were identified, one that actively interacted with T cells and B cells and was enriched in cirrhotic blood compared with HCC blood samples. Decreased naïve T cell infiltration and increased neutrophil infiltration in the liver may indicate the development of HCC in cirrhotic patients. Alterations in blood-resident immune cells may also be a sign of HCC development in cirrhotic patients. The dynamics of the immune cell subsets may serve as novel biomarkers to predict the transition from cirrhosis to HCC.

Perfluoroethylcyclohexane sulphonate, an emerging perfluoroalkyl substance, disrupts mitochondrial membranes and the expression of key molecular targets in vitro

Perfluoroethylcyclohexane sulphonate (PFECHS) is an emerging, replacement perfluoroalkyl substance (PFAS) with little information available on the toxic effects or potencies with which to characterize its potential impacts on aquatic environments. This study aimed to characterize effects of PFECHS using in vitro systems, including rainbow trout liver cells (RTL-W1 cell line) and lymphocytes separated from whole blood. It was determined that exposure to PFECHS caused minor acute toxic effects for most endpoints and that little PFECHS was concentrated into cells with a mean in vitro bioconcentration factor of 81 ± 25 L/kg. However, PFECHS was observed to affect the mitochondrial membrane and key molecular receptors, such as the peroxisome proliferator receptor, cytochrome p450-dependent monooxygenases, and receptors involved in oxidative stress. Also, glutathione-S-transferase was significantly down-regulated at a near environmentally relevant exposure concentration of 400 ng/L. These results are the first to report bioconcentration of PFECHS, as well as its effects on the peroxisome proliferator and glutathione-S-transferase receptors, suggesting that even with little bioconcentration, PFECHS has potential to cause adverse effects.

The imbalance of liver resident macrophages polarization promotes chronic autoimmune hepatitis development in mice.

Autoimmune hepatitis (AIH) is a chronic immune-mediated inflammatory liver disease. At present, it is largely unknown how the innate immune cells influence AIH development. To inquiry about mechanism of liver resident macrophages in AIH development, thus offering a new direction for AIH targeted treatment. The liver resident macrophages were eliminated by clodronate liposomes in AIH liver tissues, followed by HE and Picrosirius assay to detect liver fibrosis and lymphocyte infiltration. The liver resident macrophages polarization was detected by Immunohistochemistry and qPCR. The collagenase digestion was used to isolate Kupffer cells from AIH mice liver tissues and pro-/anti-inflammatory cytokines were determined by qPCR. M2 macrophages were the dominant phenotype at early immune response stage and hepatic inflammation was progressively aggravated after depletion of liver resident macrophages. M2 macrophages could effectively delay the development of AIH and could be polarized to M1 macrophages at the disease progresses. TLR2 ligands could promote M2 macrophages producing anti-inflammatory cytokines, whereas TLR4 ligands could promote M1 macrophages producing proinflammatory cytokines. The change of TLR2 and TLR4 ligands could lead to continuous high expression of TLR4 and decreased expression of TLR2 in macrophages to further affect liver resident macrophages polarization state. TLR2 and TLR4 ligands mediated liver resident macrophages polarization to favor chronic autoimmune hepatitis development.

Correlation of Serum Chemokine Ligand 14 with Barcelona Clinic Liver Cancer Stage, Lymphocyte Profile, and Response to Transarterial Chemoembolization in Patients with Hepatocellular Carcinoma

PurposeTo investigate the clinical relevance of serum chemokine ligand 14 (sCCL14) in patients with hepatocellular carcinoma (HCC) and the effect of transarterial chemoembolization (TACE) on the expression level of sCCL14 and the immune microenvironment. Materials and MethodsIn this prospective single-center observational study, 52 patients with HCC were recruited from January 2019 to December 2021, their clinical data and blood samples were collected, and the relationship between sCCL14 and progression-free survival (PFS) and TACE treatment response was analyzed. ResultsAmong the 52 patients with HCC (Barcelona Clinic Liver Cancer [BCLC] Stage A, 25.0%; BCLC Stage B, 44.2%; and BCLC Stage C, 30.8%), patients with BCLC Stage C HCC had significantly lower sCCL14 levels than those of patients with BCLC Stages A and B HCC (P = .001). sCCL14 levels were significantly higher in the first week after treatment than before TACE treatment (P = .024). Baseline sCCL14 levels in patients who showed complete response after TACE treatment were significantly higher than those in other groups, and lower baseline sCCL14 values were associated with shorter PFS times. Multivariate Cox regression analysis showed that sCCL14 level (hazard ratio, 1.855; 95% CI, 1.039–3.311; P = .037) was an independent prognostic factor of PFS. sCCL14 levels negatively correlated with the proportion of B lymphocytes and regulatory T cells in circulating blood and positively correlated with the absolute T-lymphocyte count. ConclusionssCCL14 may be a predictive biomarker of TACE effectiveness. Further studies are needed to validate and outline the role of combination immunotherapy.

Liver Tumor Markers, HALP Score, and NLR: Simple, Cost-Effective, Easily Accessible Indexes for Predicting Prognosis in ICC Patients after Surgery.

To investigate the prognostic significance of liver tumor markers, the hemoglobin, albumin, lymphocyte, and platelet (HALP) score; neutrophil-to-lymphocyte ratio (NLR); and platelet-to-lymphocyte ratio (PLR), for predicting the specific site of recurrence or metastasis after surgery in patients with intrahepatic cholangiocarcinoma (ICC). In total, 162 patients with pathologically proven ICC who underwent curative surgery at Sun Yat-sen University Cancer Center between April 2016 and April 2020 were analyzed. Clinicopathological characteristics were collected retrospectively. The Kaplan-Meier method was used to analyze the overall survival (OS) and recurrence-free survival (RFS). Significant clinical factors were examined by univariate analysis and multivariate analysis and analyzed by receiver operating characteristic (ROC) curve analysis. The cutoff values for the HALP score, NLR, and PLR were determined to be 43.63, 3.73, and 76.51, respectively, using the surv_cutpoint function of survminer using RFS as the target variable. In multivariate analysis, vascular invasion, pathology nerve tract invasion, and carbohydrate antigen 19-9 (CA19-9) levels were independent prognostic factors of OS, whereas the tumor number, pathology microvascular invasion, pathology differentiation, CA19-9 levels, and NLR were independent prognostic factors of RFS. For the whole recurrence analysis, the carcinoembryonic antigen (CEA) index exhibited the largest ROC curve area of all (AUC = 0.590), and the alpha-fetoprotein (AFP) index exhibited the smallest ROC curve area (AUC = 0.530). The HALP score exhibited the largest ROC curve area of all in predicting intrahepatic recurrence (AUC = 0.588), the NLR showed the best predictive value in predicting lymph node metastasis (AUC = 0.703), and the AUC of the CA19-9 index was the largest of all variables in predicting distant metastasis (AUC = 0.619). Our study showed that CA19-9, CEA, HALP score, and NLR are easily accessible, reliable, cost-effective indexes for predicting the specific site of recurrence or metastasis after surgery in ICC patients. Patients with high HALP scores and NLR have a higher risk of intrahepatic and lymph node metastasis recurrence.

Do Semaphorins Play a Role in Development of Fibrosis in Patients with Nonalcoholic Fatty Liver Disease?

Nonalcoholic fatty liver disease (NAFLD) is associated with systemic changes in immune response linked with chronic low-grade inflammation and disease progression. Semaphorins, a large family of biological response modifiers, were recently recognized as one of the key regulators of immune responses, possibly also associated with chronic liver diseases. The aim of this study was to identify semaphorins associated with NAFLD and their relationship with steatosis and fibrosis stages. In this prospective, case-control study, serum semaphorin concentrations (SEMA3A, -3C, -4A, -4D, -5A and -7A) were measured in 95 NAFLD patients and 35 healthy controls. Significantly higher concentrations of SEMA3A, -3C and -4D and lower concentrations of SEAMA5A and -7A were found in NAFLD. While there was no difference according to steatosis grades, SEMA3C and SEMA4D significantly increased and SEMA3A significantly decreased with fibrosis stages and had better accuracy in predicting fibrosis compared to the FIB-4 score. Immunohistochemistry confirmed higher expression of SEMA4D in hepatocytes, endothelial cells and lymphocytes in NAFLD livers. The SEMA5A rs1319222 TT genotype was more frequent in the NAFLD group and was associated with higher liver stiffness measurements. In conclusion, we provide the first evidence of the association of semaphorins with fibrosis in patients with NAFLD.

Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

IntroductionThe on-target off-tumor toxicity of chimeric antigen receptor-engineered T cells (CAR-T) might lead to fatal side effects in cancer patients, which remains as a major obstacle to the clinical application of CAR-T immunotherapy. The off-tumor on-target normal tissue toxicity of CAR-T cells needs to be evaluated in preclinical studies using rational animal models. ObjectivesWe aim to develop a rational animal model for assessing the off-tumor on-target normal tissue toxicity of various CAR-T cell designs quickly. MethodsWe used a recombinant adenovirus type 5 carrying human HER2/ERBB2 (Ad5-HER2) or CD47 gene (Ad5-CD47) to rapidly generate a mouse model with tunable human antigen expression on normal liver tissue to determine immunotoxicity of traditional CAR-T and hypoxia-response CAR-T cells in vivo. ResultsThe obvious liver damage and lymphocyte infiltration were not observed in mice with human antigen-high livers 8 days post-infection. Interestingly, the lethal liver damage, systemic cytokine release and CAR-T cells infiltration in liver were only observed in mice that received traditional CAR-T cells, but not in hypoxia-response CAR-T cells. ConclusionAdenovirus-based expression of target antigen in normal mouse tissue may be a useful method for assessing on-target CAR-T cell toxicity in normal tissues, especially various CAR-T cell designs that have the potency of conditional regulation in tumor microenvironment (TME).

High Number and Specific Comorbidities Could Impact the Immune Response in COVID-19 Patients.

BackgroundCellular immunodeficiency and comorbidities are common in COVID-19 patients.AimThe purpose of this study was to investigate comorbidities impacting on the cellular immunity in COVID-19 patients.MethodsThe research objects included 55 healthy controls and 718 COVID-19 patients who divided into the control group and the COVID-19 group, respectively. Those in the COVID-19 group were divided into subgroups on the basis of the number and types of comorbidities present. Lymphocyte itself and its subsets were compared between the control group and the COVID-19 group, the groups with comorbidities based on the different number and types of comorbidities, and the relationship between the lymphocyte counts and subsets with the number and types of comorbidities was investigated.ResultsCompared with the control group, the lymphocyte counts and T cell subsets were significantly increased in the groups with comorbidities, but both B and NK cell subsets were significantly decreased in the no comorbidity group and in most of the groups with comorbidities (all P<0.05). In the three comorbidities group, the lymphocyte counts and T cell subsets were all significantly decreased, but the CD56+ percentage was obviously increased (all P<0.05). The number of comorbidities was negatively correlated with the lymphocyte counts and the T and NK cell subsets. A negative correlation also existed between cancer and both the lymphocyte counts and the T cell subsets, between chronic hepatitis B and the lymphocyte counts, and between chronic kidney disease and the CD3+ counts. A positive correlation existed between nonalcoholic fatty liver disease (NAFLD) disease and both lymphocyte and CD3+ counts. The risk factors were number of comorbidities for the lymphocyte count, CD3+CD4+ and CD3+CD8+ percentages, NAFLD for the lymphocyte and CD3+ counts, cardiovascular diseases for CD3+CD4+ and CD3+CD8+ percentages, diabetes mellitus for the CD3+CD8+ percentage, and cancer for the CD3+ percentage, respectively.ConclusionsHigh numbers of comorbidities and specific comorbidities could impact the immune response of COVID-19 patients. This study provides a reference for clinicians in the identification of suitable and timely immunotherapy for COVID-19 patients.Clinical Trial Registryhttps://www.chictr.org.cn/enindex.aspx, identifier ChiCTR2000034563.

FOXO1 Alleviates Liver Ischemia-reperfusion Injury by Regulating the Th17/Treg Ratio through the AKT/Stat3/FOXO1 Pathway.

Hepatic ischemic reperfusion injury (IRI) occurring during surgery seriously affects patient prognosis. The specific mechanism of IRI has not been fully elucidated. The study aim was to explore the changes of inflammatory environment, and the relationship of the Th17/Treg cell ratio and FOXO1 expression in hepatic IRI. Liver samples at different ischemic times were collected from patients and mice. The expression of inflammatory markers and FOXO1 in the liver was detected by western blotting and qPCR. Phenotypic changes of liver lymphocytes were analyzed by flow cytometry. The AKT/Stat3/FOXO1 pathway was verified by targeting AKT with GSK2141795. The role of FOXO1 in liver inflammation and changes in lymphocyte phenotype was confirmed by upregulating FOXO1 with resveratrol. Prolonged ischemic time aggravates liver injury in both humans and mouse models of hepatic IRI. IR-stress caused Th17/Treg imbalance and FOXO1 down-regulation by activating the AKT/Stat3/FOXO1 signaling pathway. Upregulation of FOXO1 reversed the Th17/Treg cytokine imbalance and altered the inflammation environment in the liver. Liver IRI induced Th17/Treg imbalance. Upregulation of FOXO1 reversed the imbalance and alleviated liver inflammation.

Decrease of spleen invariant natural killer T and natural killer cell numbers in Burkholderia thailandensis-infected C57BL/6 mice.

Burkholderia thailandensis is a non-pathogenic bacterium that is closely related to B. pseudomallei. Invariant natural killer T (iNKT) cells are innate lymphoid cells that play a role in bacterial infections; however the iNKT cells in B. thailandensis infections are still uncharacterized. To study the cytokine production in α-galactosylceramide (α-GalCer)-stimulated lymphocytes from mouse organs. The numbers of spleen iNKT cells, natural killer (NK) cells, dendritic cells and macrophages in B. thailandensis- infected C57BL/6 (B6) mice were investigated. Lymphocytes, obtained from mouse lungs, liver, and spleen, were cultured for 48 hours with α-GalCer, and their cytokine levels were determined. iNKT, dendritic, macrophage and NK cells in the spleen of B. thailandensis-infected B6 mice or iNKT knock out (KO) mice, stimulated with either phosphate-buffered saline (PBS) or α-GalCer, were analyzed by flow cytometry. This was also done in adoptive cell transfer experiments. Interferon gamma (IFN-γ) was predominantly produced in α-GalCer-stimulated mouse spleen and liver lymphocytes, while interleukin (IL)-13 was the main cytokine found in the lungs. B. thailandensis-infected mice had a significantly lower number of splenic iNKT, NK and dendritic cells, but not macrophages, compared to the control. Interestingly, the number of NK cells was significantly decreased in iNKT wild type and iNKT KO mice after B. thailandensis infection. The number of NK cells recovered by activation with α-GalCer or after adoptive transfer of iNKT cells into KO mice. The iNKT cell-mediated reduction of dendritic and NK cells might be related to infection by B. thailandensis. B. thailandensis decreased the number of iNKT and NK cells in the spleen of infected mice.

Activated Natural Killer Cell Promotes Nonalcoholic Steatohepatitis Through Mediating JAK/STAT Pathway.

Background & AimsHepatic immune microenvironment plays a pivotal role in the development of nonalcoholic steatohepatitis (NASH). However, the role of natural killer (NK) cells, accounting for 10%–20% of liver lymphocytes, in NASH is still unclear. In this study, we aim to investigate the functional significance of NK cells in NASH evolution.MethodsNASH was induced in mice fed methionine- and choline-deficient diet (MCD), choline-deficient high-fat diet (CD-HFD), or high-fat diet with streptozotocin injection (STAM model). NK cell deficient mice (Nfil3-/-) and neutralization antibody (PK136) were used in this study.ResultsActivated liver NK cells were identified with increased expression of NKG2D, CD107a, and interferon-γ but decreased inhibitory NKG2A. With NK cell deficiency Nfil3-/- mice, the absence of NK cells ameliorated both MCD- and CDHF- induced NASH development with significantly decreased hepatic triglycerides, peroxides, alanine aminotransferase, and aspartate aminotransferase compared with Nfil3+/+ mice. Further molecular analysis unveiled suppressed pro-inflammatory cytokines and associated signaling. Mechanistically, NK cells isolated from NASH liver secreted higher levels of pro-inflammatory cytokines (interferon-γ, interleukin 1β, interleukin 12, CCL4, CCL5, and granulocyte-macrophage colony-stimulating factor), which could activate hepatic JAK-STAT1/3 and nuclear factor kappa B signaling and induce hepatocyte damage evidenced by elevated reactive oxygen species and apoptosis rate. Moreover, neutralization antibody PK136-dependent NK cell depletion can significantly alleviate MCD-induced steatohepatitis with suppressed cytokine levels and JAK-STAT1/3 activity.ConclusionsNK cells in NASH liver are activated with a more pro-inflammatory cytokine milieu and promote NASH development via cytokine-JAK-STAT1/3 axis. Modulation of NK cells provides a potential therapeutic strategy for NASH.

Abstract 120: Compartmentalized functions of epidermal growth factor receptor (EGFR) in tumorigenesis and malignant phenotypes

Abstract Previous studies demonstrated that nuclear EGFR is associated with malignancy and resistance to tyrosine kinase inhibitor (TKI). To decipher the cellular compartmentalized functions and underlying mechanism of EGFR, we generated EGFR mutants at nuclear translocation signal (NLS) and nuclear export signal (NES). The results showed that EGFR NLS mutant with decreased nuclear translocation sensitized the cancer cells to TKI and impaired invasiveness and stemness of the cancer cells. In contrast, EGFR NES mutant with increased nuclear accumulation promoted aggressiveness of the cancer cells including TKI resistance. Next, we generated genetically engineered mouse model (GEMM) to validate the resulting phenotypes of the EGFR mutants. To this end, CRISPR-Cas9-mediated genome editing approach was used for generating EGFR mutant knock-in mouse, which harbors an internalization-deficient mutation (EGFRLL/AA), pro-nuclear (NES) mutation (EGFRL749P(S)) and the dual mutations. Upon EGF stimulation nuclear EGFR (nEGFR) accumulation in the hepatocytes from EGFRL749P heterozygous mice was increased. Conversely, a significant reduction of nEGFR was observed in the hepatocyte from EGFTRLL/AA homozygous mice. We found that germline expression of EGFRL749P(S) gave rise to deficiency in breeding. However, EGFRLL/AA mutant mice displayed no gross abnormal even in homozygous offspring, suggesting that depletion of nEGFR has no significant phenotype. We failed to generate homozygotes of EGFR NES mutant mice due to embryonic lethality. Of note, the female and male founders and F1 EGFRL798P(S) mice developed tumors with huge spleen. The IHC staining results indicated that the infiltrating lymphocytes in liver, stomach and kidney were B cells. The latency of the B cell lymphomagenesis was from 17 to 23 months, and tumor incidence of F1 offspring of EGFRL749P(S) knock-in mice was 10 of 15 (66.7%). These findings indicated that single allele mutation in EGFR NES domain is capable of driving lymphomagenesis, suggesting that the EGFR NES mutant possesses a strong oncogenic activity. Surprisingly, by searching database of EGFR mutation in human tumors (Cosmic database https://cancer.sanger.ac.uk/cosmic ), we noticed that among the somatic mutations in EGFR gene the gene amplification represents only small part of the gene alternations in human cancers, and EGFR L747P and L747S mutation occur in the lung cancer patients with intrinsic TKI resistance. In addition, the majority of human B lymphocyte originated tumors harboring EGFR NES mutation are also heterozygotes. Cosmic database shows that somatic mutations at the EGFR NES region occur in the patients with plasma cell lymphoma. Taken together, our findings suggest that nEGFR contributes to malignant potential of the tumors and EGFR with NES mutation is a tumor driver. Citation Format: Lei Nie, Junwei Hou, Yu-Yi Chu, Ying-Nai Wnag, Li-Chuan Chan, Longfei Huo, Paul Chiao, Shao-Chun Wang, Mien-Chie Hung, Michael Wang. Compartmentalized functions of epidermal growth factor receptor (EGFR) in tumorigenesis and malignant phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 120.

Suppression of systemic inflammation and signs of acute and chronic cholangitis by multi-kinase inhibitor 1-(4-Cl-benzyl)-3-chloro-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione.

An aberrant activity of growth factor receptors followed by excessive cell proliferation plays a significant role in pathogenesis of cholangitis. Therefore, inhibition of these processes could be a fruitful therapeutic strategy. The effects of multi-kinase inhibitor 1-(4-Cl-benzyl)-3-chloro-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione (MI-1) on the hepatic and systemic manifestations of acute and chronic cholangitis in rats were addressed. MI-1 (2.7mg/kg per day) was applied to male rats that experienced α-naphthylisothiocyanate-induced acute (3days) or chronic (28days) cholangitis. Liver autopsy samples, blood serum markers, and leukograms were studied. MI-1 localization in liver cells and its impact on viability of HepG2 (human hepatoma), HL60 (human leukemia), and NIH3T3 (normal murine fibroblasts) cell lines and lymphocytes of human peripheral blood (MTT, DNA fragmentation, DNA comet assays, Propidium Iodide staining) were assessed. Under both acute and chronic cholangitis, MI-1 substantially reduced liver injury, fibrosis, and inflammatory scores (by 46-86%) and normalized blood serum markers and leukograms. Moreover, these effects were preserved after a 28-day recovery period (without any treatment). MI-1 inhibited the HL60, HepG2 cells, and human lymphocytes viability (IC50 0.6, 9.5 and 8.3µg/ml, respectively), while NIH3T3 cells were resistant to that. Additionally, HepG2 cells and lymphocytes being incubated with MI-1 demonstrated insignificant pro-apoptotic and pro-necrotic changes and DNA single-strand breaks, suggesting that MI-1 effects in liver might be partly caused by its cytotoxic action towards liver cells and lymphocytes. In conclusion, MI-1 attenuated the systemic inflammation and signs of acute and chronic cholangitis partly through cytotoxicity towards cells of hepatic and leukocytic origin.

Analyzing sensitivity of the energy metabolism in the tissues of the heart, liver, kidney, and blood lymphocytes in rats to the effect of local vibration and pharmacological protection by a succinate-containing antihypoxanth

Introduction. Modern technological processes that generate ultra- and infrasound, electromagnetic, vibration-noise radiation significantly exceed the background effects and inevitably create an unfavorable technogenic load on the human body. A special place is occupied by vibration, primarily associated with such professional groups as mining enterprises, gold and diamond processing plants, aircraft plants, thermal power plants and hydroelectric power plants, and aluminum industry enterprises. Vibration exposure leads to a number of biochemical disorders in the homeostasis system. Mitochondrial dysfunctions are among the leading elements in the hierarchy of pathogenesis of many pathological syndromes and diseases. The aim of the study was an experimental analysis of tissue-specific features of the myocardium, kidneys, liver energy production system activity as well as the enzyme status of rat lymphocytes in relation to the activity of succinate dehydrogenase (SDH) on the background of 7 episodes of local vibration with a frequency of 27-30 Hz for 90 minutes daily before and after protection with succinate-containing antihypoxant "Yantar-cardio" at a dose of 100 mg/kg/day. Materials and methods. The study of the energy-dependent reactions of the native mitochondria of the organs was carried out by the polarographic method using a closed Clark-type membrane electrode. The activity of SDH of rat blood lymphocytes was studied by a modified method of quantitative cytochemical analysis. Results. Functional rearrangement in the respiratory chain (RC) of the mitochondria confirmed the NAD-dependent site's inhibition and activation of the oxidation system of endogenous succinic acid, most pronounced in the myocardial tissue. On the vibration background, the specific SDH activity of lymphocytes (Q) significantly increased by 52%, and the variability (V) - by three times. The use of "Yantar-cardio" limited the growth rate of endogenous respiration of the most significant in the heart (70%, p&lt;0.05), restored oxidative and conjugating activity NAD-dependent link of RC, improve the structure and condition of the cell population of lymphocytes based on their energy status and warned the mismatch of the leading parameters of the population variability of the cellular pool of blood lymphocytes in experimental animals. Conclusions. The intensity of vibration-mediated bioenergetic hypoxia in the form of inhibition of the NAD-dependent link of the respiratory chain and activation of the endogenous succinic acid oxidation system is tissue-specific and is most pronounced in the myocardial tissue. The succinic acid oxidation system is highly sensitive to vibration effects and a pharmacological drug that supports the function of RC under conditions of the stress of the homeostatic functions of tissues involved in the pathological process.

From the Editor's Desk...

From the Editor's Desk...