Chemosensory protein 22 in Riptortus pedestris is involved in the recognition of three soybean volatiles.

Riptortus pedestris (Hemiptera: Alydidae), a common agricultural pest, is the major causative agent of "soybean staygreen." However, the interactions between chemosensory proteins (CSPs) in R. pedestris and host plant volatiles have yet to be comprehensively studied. In this study, we performed real-time fluorescence quantitative polymerase chain reaction (PCR) to analyze the antennal expression of RpedCSP22 and subsequently analyzed the interactions between 21 soybean volatiles, five aggregation pheromones, and RpedCSP22 protein in vitro using a protein expression system, molecular docking, site-directed mutagenesis, and fluorescence competitive binding experiments. The RpedCSP22 protein showed binding affinity to three soybean volatiles (benzaldehyde, 4-ethylbenzaldehyde, and 1-octene-3-ol), with optimal binding observed under neutral pH conditions, and lost binding ability after site-directed mutagenesis. In subsequent RNA interference (RNAi) studies, gene silencing was more than 90 %, and in silenced insects, electroantennographic responses were reduced by more than 75 % compared to non-silenced insects. Moreover, Y-tube olfactory behavioral assessments revealed that the attraction of R. pedestris to the three soybean volatiles was significantly attenuated. These findings suggest that RpedCSP22 plays an important role in the recognition of host plant volatiles by R. pedestris andprovides a theoretical basis for the development of novel inhibitors targeting pest behavior.

Time-Dependent Sequential Changes of IL-10 and TGF-ß1 in Mice with Deep Vein Thrombosis.

OBJECTIVES: To detect the expression changes of interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1) during the development of deep vein thrombosis in mice, and to explore the application value of them in thrombus age estimation. METHODS: The mice in the experimental group were subjected to ligation of inferior vena cava. The mice were sacrificed by excessive anesthesia at 1 d, 3 d, 5 d, 7 d, 10 d, 14 d and 21 d after ligation, respectively. The inferior vena cava segment with thrombosis was extracted below the ligation point. The mice in the control group were not ligated, and the inferior vena cava segment at the same position as the experimental group was extracted. The expression changes of IL-10 and TGF-ß1 were detected by immunohistochemistry (IHC), Western blotting and real-time qPCR. RESULTS: IHC results revealed that IL-10 was mainly expressed in monocytes in thrombosis and TGF-ß1 was mainly expressed in monocytes and fibroblast-like cells in thrombosis. Western blotting and real-time qPCR showed that the relative expression levels of IL-10 and TGF-ß1 in each experimental group were higher than those in the control group. The mRNA and protein levels of IL-10 reached the peak at 7 d and 10 d after ligation, respectively. The mRNA expression level at 7 d after ligation was 4.72±0.15 times that of the control group, and the protein expression level at 10 d after ligation was 7.15±0.28 times that of the control group. The mRNA and protein levels of TGF-ß1 reached the peak at 10 d and 14 d after ligation, respectively. The mRNA expression level at 10 d after ligation was 2.58±0.14 times that of the control group, and the protein expression level at 14 d after ligation was 4.34±0.19 times that of the control group. CONCLUSIONS: The expressions of IL-10 and TGF-ß1 during the evolution of deep vein thrombosis present time-dependent sequential changes, and the expression levels of IL-10 and TGF-ß1 can provide a reference basis for thrombus age estimation.

A ratiometric fluorescent probe for fast detection and bioimaging of formaldehyde.

A novel ratiometric probe (SWJT-10) based on isophorone derivatives has been designed and synthesized for the detection of formaldehyde (FA). This probe displayed an obvious ratiometric fluorescence response to FA with a blue shift from the NIR (680 nm) to the yellow light region (600 nm) in aqueous solution. And it showed good selectivity, high sensitivity and a fast response to FA (less than 5 s) due to a new recognition mechanism. Moreover, SWJT-10 has been applied to monitor FA in living cells and zebrafish.

Symptom burden survey and symptom clusters in patients with cervical cancer: a cross-sectional survey.

PURPOSE: The purpose of this study is to determine the incidence and severity of symptoms of patients with cervical cancer within 6 months after radiotherapy and chemotherapy, form a symptom burden report, evaluate the distribution characteristics of symptoms, identify symptom clusters, and provide a basis for clinical doctors and nurses to improve the symptom management of patients with cervical cancer after radiotherapy and chemotherapy. METHODS: The patients with cervical cancer within 6 months after radiotherapy and chemotherapy were recruited to investigate their symptom burden. Exploratory factor analysis was used to identify symptom clusters. RESULTS: A total of 250 patients participated in the study. The study found that the most common symptom among the 40 symptoms was fatigue, and the most serious symptom was nocturia. Based on the occurrence rate and severity of symptoms, nine symptom clusters were identified, including psycho-emotion-related symptom cluster, pain-disturbed sleep-related symptom cluster, menopausal symptom cluster, tinnitus-dizziness-related symptom cluster, urinary-related symptom cluster, dry mouth-bitter taste-related symptom cluster, intestinal-related symptom cluster, memory loss-numbness-related symptom cluster, and emaciation-related symptom cluster. The three most serious symptom clusters are pain-disturbed sleep-related symptom cluster, urinary-related symptom cluster, and memory loss-numbness-related symptom cluster. CONCLUSION: The symptoms of patients with cervical cancer within 6 months after radiotherapy and chemotherapy are complex, and nine symptom clusters can be identified according to the incidence and severity of symptoms. We can find the potential biological mechanism of each symptom cluster through the discussion of previous mechanism research and clinical research. The number of symptom clusters and the number of symptoms within the symptom cluster are closely related to the symptom evaluation scale selected for the study. Therefore, the symptom cluster study urgently needs a targeted symptom evaluation scale that can comprehensively reflect the patient's condition.

Replication study and meta-analysis indicate a suggestive association of RUNX3 locus with primary biliary cholangitis.

Susceptibility to primary biliary cholangitis (PBC) is in part genetically determined. In our previous PBC genome-wide association study (GWAS) in 1118 Han Chinese PBC and 4036 controls, we noted that multiple SNPs in the runt-related transcription factor 3 (RUNX3) regions showed a nominally significant association. The tag SNP rs7529070 was genotyped using a TaqMan assay in a separately collected 1435 PBC and 3205 controls. A meta-analysis with a combined 2553 PBC and 7241 controls showed that rs7529070 is still nominally associated with PBC (p = 1.7 × 10-4, odds ratio (OR) = 1.18, 95% confidence interval (CI) = 1.08-1.28). Further analysis indicated that the risk allele of rs7529070 (G allele) is in complete linkage disequilibrium (LD) (r2 = 1) with the G allele of rs4648889, which is known to be associated with increased RUNX3 expression. Bioinformatic analysis with existing expression data showed that the expression of RUNX3 is significantly increased in PBC patients (p = 0.001) and the expression level is correlated with disease severity. Consistently, we also found significantly increased RUNX3 expression (p < 0.01) in the livers of dnTGFßRII mice (a PBC mouse model). This study suggests that the RUNX3 locus may associate with PBC in Han Chinese.

Influence of Metabolic Risk Factors on Risk of Hepatocellular Carcinoma and Liver-Related Death in Men With Chronic Hepatitis B: A Large Cohort Study.

BACKGROUND & AIMS: Little is known about the absolute risk of hepatocellular carcinoma (HCC) and liver-disease related death, in association with metabolic risk factors, for patients with hepatitis B virus (HBV) infection. METHODS: We collected data from 5373 male Taiwanese civil servants who visited Taiwan's Government Employees' Central Clinics and received routine free physical examinations from 1989 through 1992. We obtained information on liver-related morbidity and mortality in HBV carriers, 40-65 years of age (n=1690), with different metabolic risk factors. We compared their medical histories with those of study participants without HBV or HCV infection in the same age range (n=1289). We used patients' baseline data on obesity, diabetes, hypertriglyceridemia, and high blood pressure to assign them to metabolic risk categories. We then performed a case-cohort analysis of the effects of hepatitis B viral factors on risk for HCC, based on metabolic factors and insulin resistance. RESULTS: Over a median follow-up period of 19 years, 158 of the 1690 HBV carriers developed HCC and 126 died from liver-related diseases. Among participants without HBV or HCV infection, only 6 developed HCC or died from liver-related disease. HBV carriers with different metabolic risk factors had significant differences in cumulative incidence of HCC and liver-related death. Patients with 3 or more metabolic risk factors had a substantially higher risk for HCC (10-year cumulative incidence, 13.60%) than patients with a low metabolic risk profile (10-year cumulative incidence, 4.83%; adjusted-hazard ratio, 2.32; 95% CI, 1.18-4.54). Smoking had a significant effect on this association (Pinteraction = .0044). Having 3 or more metabolic risk factors, compared with no factors, significantly increased the risk of HCC (adjusted-hazard ratio, 5.06; 95% CI, 2.23-11.47) and 10-year cumulative incidence of HCC (25.0% in smokers with 3 or more metabolic risk factors vs 3.87% in smokers with none; P < .0001) in smokers, but did not increase risk of HCC in nonsmokers. Metabolic risk factors and insulin resistance had the largest effect on HCC risk in patients with levels of HBV-DNA <10,000 copies/mL. CONCLUSIONS: In a study of men with chronic HBV infection ages 40-65 years in Taiwan, we associated a high burden of metabolic risk factors with increased risk of HCC; smoking has a significant effect on this association.

CD8+ T cells and IFN-γ induce autoimmune myelofibrosis in mice.

Myelofibrosis usually occurs either as a part of a myelodysplastic syndrome or in conjunction with neoplasia. It is not commonly thought of an autoimmune disease. We reported that p40-/-IL-2Rα-/- (interleukin-12p40 and interleukin-2 receptor alpha double knockout) mice, a mouse model of human primary biliary cholangitis, exhibited features consistent with autoimmune myelofibrosis, including anemia associated with bone marrow fibrosis, and extramedullary hematopoiesis (EMH) including LSK (Lineage-c-Kit+Sca-1+) cells in spleen, liver and peripheral blood. There were also increased LSK cells in bone marrow but they demonstrated impaired hematopoiesis. Importantly effector memory T cells that infiltrated the bone marrow of p40-/-IL-2Rα-/- mice manifested a higher ability to produce IFN-γ. CD8+ T cells, already known to play a dominate role in portal inflammation, were also key for bone marrow dysregulation and EMH. IFN-γ was the key cytokine that induced bone marrow fibrosis, bone marrow failure and EMH. Finally anti-CD8α antibody therapy fully protected p40-/-IL-2Rα-/- mice from autoimmune myelofibrosis. In conclusion, our results demonstrate that CD8+ T cells and IFN-γ are associated with autoimmune myelofibrosis, a finding that may allow targeting of CD8+ T cells and IFN-γ as a therapeutic targets.

The molecular basis of immune regulation in autoimmunity.

Autoimmune diseases can be triggered and modulated by various molecular and cellular characteristics. The mechanisms of autoimmunity and the pathogenesis of autoimmune diseases have been investigated for several decades. It is well accepted that autoimmunity is caused by dysregulated/dysfunctional immune susceptible genes and environmental factors. There are multiple physiological mechanisms that regulate and control self-reactivity, but which can also lead to tolerance breakdown when in defect. The majority of autoreactive T or B cells are eliminated during the development of central tolerance by negative selection. Regulatory cells such as Tregs (regulatory T) and MSCs (mesenchymal stem cells), and molecules such as CTLA-4 (cytotoxic T-lymphocyte associated antigen 4) and IL (interleukin) 10 (IL-10), help to eliminate autoreactive cells that escaped to the periphery in order to prevent development of autoimmunity. Knowledge of the molecular basis of immune regulation is needed to further our understanding of the underlying mechanisms of loss of tolerance in autoimmune diseases and pave the way for the development of more effective, specific, and safer therapeutic interventions.

Genome-wide identification of blood DNA methylation patterns associated with early-onset hepatocellular carcinoma development in hepatitis B carriers.

The etiology of early-onset hepatocellular carcinoma (HCC) among hepatitis B virus (HBV) carriers remains unclear. DNA methylation levels in peripheral leukocytes have been associated with different environmental exposures and immune or inflammatory response. We aimed to identify methylation signatures of peripheral leukocytes that could track hepatitis B progression to HCC, especially for early-onset HCC. We first performed an epigenome-wide association analysis on 48 matched case-control pairs in a nested case-control study within a 22-yr follow-up cohort of HBV carriers. Through this analysis we found that progression to early-onset HCC involved methylation variable positions across the genome, in which a substantial proportion displayed significant variation due to HBV viral load, chronic hepatitis status, and/or leukocyte subtype composition, and these associations were significantly enriched among genes in immune pathways. Methylation at probes cg00300879, cg06872964, and cg07080864, that are located within the proximal promoter of CNKSR1, IFI44L, and PENK, respectively, was validated by bisulfite pyrosequencing and findings were replicated in a case-sibling study of early-onset HCC (134 cases vs. 174 sibling controls). Furthermore, a high methylation score, constructed using the three probes, was predictive for the risk of early-onset HCC in two datasets (adjusted-odds ratios = 0.21-0.32, P ≤ 0.0206). This association was also observed for late-onset HCC (adjusted-odds ratio = 0.42-0.47, P ≤ 0.0194) in a nested case-control study (120 cases vs. 178 controls). In prospective analysis, change in the score was detected 5-9 yr before HCC onset. Blood-based methylation profiling provides new insights into the complexity of virus-host interaction underlying HBV-related HCC, holding promise for the disease risk management. © 2016 Wiley Periodicals, Inc.

Characteristics of beat-to-beat photoplethysmography waveform indexes in subjects with metabolic syndrome.

UNLABELLED: Metabolic syndrome (MetS) increases the risk of the subsequent development of cardiovascular disease. This study aimed to determine if the harmonic indexes of finger photoplethysmography (PPG) waveforms can be used to discriminate different arterial pulse transmission conditions between MetS and healthy subjects. Three-minute PPG signals were obtained in 65 subjects, who were assigned to 3 age-matched groups (MS, with no less than three MetS factors; pre-MS, with one or two MetS factors; CONTROL: with no MetS factor). FDT (foot delay time) and amplitude proportions (Cn) and their standard deviations (SDn) and coefficients of variations (CVn) were calculated for harmonics 1 to 10 of the PPG waveform. FDT was smaller in MS than in CONTROL. C1 and C2 values were significantly smaller, whereas C4-C9 values were significantly or appeared to be larger in MS than in pre-MS. Most of the SDn and CVn values were largest in MS. This study is the first to demonstrate that harmonic-analysis indexes of the beat-to-beat PPG waveform can provide information about MetS-induced changes in the arterial pulse transmission and cardiovascular regulatory activities. The present findings may therefore be useful in developing a noninvasive and easy-to-perform technique that could improve the early detection of cardiovascular diseases.

Direct synthesis of chiral 3-arylsuccinimides by rhodium-catalyzed enantioselective conjugate addition of arylboronic acids to maleimides.

Chiral rhodium catalysts comprising 2,5-diaryl- substituted bicyclo[2.2.1]diene ligands L1-L10 were utilized in the enantioselective 1,4-addition reaction of arylboronic acids to N-substituted maleimides. In the presence of 2.5 mol % of Rh(I) /L2, enantioenriched conjugate addition adducts were isolated in 72-99 % yields with 86-98 % ee. This protocol offers a convenient method to access a variety of 3-arylsuccinimides in a highly enantioselective manner. Maleimides with readily cleavable N-protecting groups were tolerated enabling the synthesis of useful synthetic intermediates. Pyrrolidine 4, a biologically active compound, and pyrrolidine 5, an ent-precursor to an HSD-1 inhibitor, were synthesized to demonstrate the utility of this method.

Complexity analysis of beat-to-beat skin-surface laser-Doppler signals in diabetic subjects.

MOTIVATIONS: This study performed skin-surface laser-Doppler flowmetry (LDF) measurements with the aim of verifying if complexity analysis applied to the beat-to-beat LDF waveform index can be used to discriminate diabetic, prediabetic, and normoglycemic subjects. METHODS: Sixty-six subjects were assigned to three age-matched groups according to the results of oral glucose tolerance tests. Beat-to-beat analysis was performed on the pulsatile LDF waveform to obtain the pulse-to-mean ratio (AD) and pulse width (PW), and then approximate-entropy (ApEn) values for their 20-minute index sequences were calculated to evaluate the signal complexity. RESULTS: AD and PW did not differ significantly among the three study groups. ApEn values of AD and PW were significantly larger and marginally larger, respectively, in the diabetic group than in the prediabetic and normoglycemic groups. CONCLUSION: These results indicate the presence of significant differences in ApEn indexes among diabetic, prediabetic, and normoglycemic subjects. The presence of increased complexity in the LDF index sequence may be partly attributed to the adaptability of the microcirculatory regulatory activities or the impairment of the homeostasis mechanism of microcirculatory-blood-flow perfusion. The present findings may be pertinent to the early detection of the diabetes-induced impairment of this perfusion.

Scutellarein Ameliorated Chondrocyte Inflammation and Osteoarthritis in Rats.

OBJECTIVE: Osteoarthritis (OA) is a degenerative joint disorder characterized by the gradual degradation of joint cartilage and local inflammation. This study aimed to investigate the anti-OA effect of scutellarein (SCU), a single-unit flavonoid compound obtained from Scutellaria barbata D. Don, in rats. METHODS: The extracted rat chondrocytes were treated with SCU and IL-1ß. The chondrocytes were divided into control group, IL-1ß group, IL-1ß+SCU 50 µmol/L group, and IL-1ß+SCU 100 µmol/L group. Morphology of rat chondrocytes was observed by toluidine blue and safranin O staining. CCK-8 method was used to detect the cytotoxicity of SCU. ELISA, qRT-PCR, Western blotting, immunofluorescence, SAß-gal staining, flow cytometry, and bioinformatics analysis were applied to evaluate the effect of SCU on rat chondrocytes under IL-1ß intervention. Additionally, anterior cruciate ligament transection (ACL-T) was used to establish a rat OA model. Histological changes were detected by safranin O/fast green, hematoxylin-eosin (HE) staining, and immunohistochemistry. RESULTS: SCU protected cartilage and exhibited anti-inflammatory effects via multiple mechanisms. Specifically, it could enhance the synthesis of extracellular matrix in cartilage cells and inhibit its degradation. In addition, SCU partially inhibited the nuclear factor kappa-B/mitogen-activated protein kinase (NF-κB/MAPK) pathway, thereby reducing inflammatory cytokine production in the joint cartilage. Furthermore, SCU significantly reduced IL-1ß-induced apoptosis and senescence in rat chondrocytes, further highlighting its potential role in OA treatment. In vivo experiments revealed that SCU (at a dose of 50 mg/kg) administered for 2 months could significantly delay the progression of cartilage damage, which was reflected in a lower Osteoarthritis Research Society International (OARSI) score, and reduced expression of matrix metalloproteinase 13 (MMP13) in cartilage. CONCLUSION: SCU is effective in the therapeutic management of OA and could serve as a potential candidate for future clinical drug therapy for OA.

MiR-3074-5p Regulates Trophoblasts Function via EIF2S1/GDF15 Pathway in Recurrent Miscarriage.

Dysfunction of extravillous trophoblasts (EVTs) might cause early pregnancy failure by interfering with embryo implantation and/or placentation. We previously reported that the villus miR-3074-5p expression level was increased, whereas the peripheral level of GDF15, a predict target gene of miR-3074-5p, was decreased in recurrent miscarriages (RM) patients, and miR-3074-5p could enhance apoptosis but reduce invasion of human extravillous trophoblast cells (EVTs). The aim of this study was to further explore roles of miR-3074-5p/GDF15 pathway in regulation of EVTs function. It was validated that GDF15 was not the direct target of miR-3074-5p, whereas EIF2S1, an upstream regulator of GDF15 maturation and secretion, was the direct target of miR-3074-5p. The villus expression levels of GDF15 and EIF2S1 were significantly decreased in RM patients. Knockdown of GDF15 expression presented inhibitory effects on proliferation, migration, and invasion of HTR8/SVneo cells. Up-regulated miR-3074-5p expression led to the significant decreased GDF15 expression in HTR8/SVneo cells, and this effect could be efficiently reversed by the overexpression of EIF2S1. Meanwhile, the suppressive effects of miR-3074-5p on proliferation, migration, and invasion of HTR8/SVneo cells could be intercepted by the treatment of recombinant human GDF15 protein. Collectively, these data suggested that miR-3074-5p could reduce GDF15 production via targeting inhibition of EIF2S1 expression, and the deficiency in GDF15 function might lead to the early pregnancy loss by attenuating proliferation and invasion of EVTs.

MNSFß promotes LPS-induced TNFα expression by increasing the localization of RC3H1 to stress granules, and the interfering peptide HEPN2 reduces TNFα production by disrupting the MNSFß-RC3H1 interaction in macrophages.

Abnormally elevated tumor necrosis factor-α (TNFα) levels at the maternal-fetal interface can lead to adverse pregnancy outcomes, including recurrent miscarriage (RM), but the mechanism underlying upregulated TNFα expression is not fully understood. We previously reported that the interaction between monoclonal nonspecific suppressor factor-ß (MNSFß) and RC3H1 upregulates TNFα expression, but the precise mechanisms are unknown. In this study, we found that MNSFß stimulated the LPS-induced TNFα expression by inactivating the promoting effect of RC3H1 on TNFα mRNA degradation rather than directly inhibiting the expression of RC3H1 in THP1-Mϕs. Mechanistically, the 81-326 aa region of the RC3H1 protein binds to the 101-133 aa region of the MNSFß protein, and MNSFß facilitated stress granules (SGs) formation and the translocation of RC3H1 to SGs by interacting with RC3H1 and fragile X mental retardation 1 (FMR1) in response to LPS-induced stress. The SGs-localization of RC3H1 reduced its inhibitory effect on TNFα expression in LPS-treated THP1-Mϕs. The designed HEPN2 peptide effectively reduced the LPS-induced expression of TNFα in THP1-Mϕs by interfering with the MNSFß-RC3H1 interaction. Treatment with the HEPN2 peptide significantly improved adverse pregnancy outcomes, including early pregnancy loss (EPL) and lower fetal weight (LFW), which are induced by LPS in mice. These data indicated that MNSFß promoted TNFα expression at least partially by increasing the localization of RC3H1 to SGs under inflammatory stimulation and that the HEPN2 peptide improved the adverse pregnancy outcomes induced by LPS in mice, suggesting that MNSFß is a potential pharmacological target for adverse pregnancy outcomes caused by abnormally increased inflammation at early pregnancy.

Survey of physicians' knowledge about pediatric nonalcoholic fatty liver disease in China.

OBJECTIVES: To evaluate physicians' awareness and knowledge towards pediatric nonalcoholic fatty liver disease (NAFLD) and their attitude toward change in nomenclature from NAFLD to metabolic dysfunction-associated fatty liver disease (MAFLD) or metabolic dysfunction-associated steatotic liver disease (MASLD) in China. METHODS: The questionnaire survey contained five parts (characteristics of the participants, epidemiology, diagnosis, management of NAFLD, and attitudes toward the nomenclature of MAFLD/MASLD). The participants included 53 hepatologists, 88 gastroenterologists (GEs), 74 endocrinologists (ENDOs), 61 primary care physicians (PCPs), and 157 pediatricians across 31 municipalities, provinces and autonomous regions of China's mainland. RESULTS: Hepatologists saw the largest number of pediatric NAFLD patients annually (median 9 [range 1-20]), with the lowest number by PCPs (even notwithstanding one patient annually). The primary sources of pediatric NAFLD knowledge were acquired via guidelines. Hepatologists had the highest total knowledge score among all five types of physicians. Approximately one-third of nonspecialists (ENDOs and PCPs) considered liver biopsy necessary for pediatric NAFLD patients, and this percentage increased to half in specialists (hepatologists and GEs). For nonspecialists, the major barriers to the management of pediatric NAFLD were poor patient adherence to lifestyle modifications and lacking confidence in managing NAFLD. Above 90% physicians agreed to change the nomenclature NAFLD to MAFLD; however, they were not sure whether it could reduce the economic burden. CONCLUSIONS: Despite the epidemic of pediatric NAFLD in China, a significant knowledge gap remains in the identification, diagnosis, and treatment of pediatric NAFLD, particularly among frontline workers such as pediatricians and PCPs. More education programs should be carried out in the future.

Lymphatic endothelial cell-mediated accumulation of CD177+Treg cells suppresses antitumor immunity in human esophageal squamous cell carcinoma.

Regulatory T (Treg) cells are critical in shaping an immunosuppressive microenvironment to favor tumor progression and resistance to therapies. However, the heterogeneity and function of Treg cells in esophageal squamous cell carcinoma (ESCC) remain underexplored. We identified CD177 as a tumor-infiltrating Treg cell marker in ESCC. Interestingly, expression levels of CD177 and PD-1 were mutually exclusive in tumor Treg cells. CD177+ Treg cells expressed high levels of IL35, in association with CD8+ T cell exhaustion, whereas PD-1+ Treg cells expressed high levels of IL10. Pan-cancer analysis revealed that CD177+ Treg cells display increased clonal expansion compared to PD-1+ and double-negative (DN) Treg cells, and CD177+ and PD-1+ Treg cells develop from the same DN Treg cell origin. Importantly, we found CD177+ Treg cell infiltration to be associated with poor overall survival and poor response to anti-PD-1 immunotherapy plus chemotherapy in ESCC patients. Finally, we found that lymphatic endothelial cells are associated with CD177+ Treg cell accumulation in ESCC tumors, which are also decreased after anti-PD-1 immunotherapy plus chemotherapy. Our work identifies CD177+ Treg cell as a tumor-specific Treg cell subset and highlights their potential value as a prognostic marker of survival and response to immunotherapy and a therapeutic target in ESCC.

Targeting pathogenic CD8+ tissue-resident T cells with chimeric antigen receptor therapy in murine autoimmune cholangitis.

Primary biliary cholangitis (PBC) is a cholestatic autoimmune liver disease characterized by autoreactive T cell response against intrahepatic small bile ducts. Here, we use Il12b-/-Il2ra-/- mice (DKO mice) as a model of autoimmune cholangitis and demonstrate that Cd8a knockout or treatment with an anti-CD8α antibody prevents/reduces biliary immunopathology. Using single-cell RNA sequencing analysis, we identified CD8+ tissue-resident memory T (Trm) cells in the livers of DKO mice, which highly express activation- and cytotoxicity-associated markers and induce apoptosis of bile duct epithelial cells. Liver CD8+ Trm cells also upregulate the expression of several immune checkpoint molecules, including PD-1. We describe the development of a chimeric antigen receptor to target PD-1-expressing CD8+ Trm cells. Treatment of DKO mice with PD-1-targeting CAR-T cells selectively depleted liver CD8+ Trm cells and alleviated autoimmune cholangitis. Our work highlights the pathogenic role of CD8+ Trm cells and the potential therapeutic usage of PD-1-targeting CAR-T cells.

The gut microbiome contributes to splenomegaly and tissue inflammation in a murine model of primary biliary cholangitis.

Background: Splenomegaly is not just a consequence of numerous chronic and acute conditions but may also contribute to their severity, due to the interaction of the spleen with the gut microbiome. This study aimed to explore the effect of the gut microbiome on splenomegaly. Methods: We used p40-/-IL-2Rα-/- mice as a murine model of primary biliary cholangitis (PBC) as per our previous study. Splenomegaly was evaluated by spleen weight. Severity of liver inflammation was evaluated by hepatic mononuclear cell (MNCs) number and pathological score. Changes of immune cells in the spleen and liver were detected by flow cytometry. The effects of the gut microbiome on splenomegaly and liver inflammation were observed by combined antibiotic treatment in p40-/-IL-2Rα-/- mice. Results: A proportion of p40-/-IL-2Rα-/- mice developed splenomegaly. The results revealed that liver mononuclear cells infiltration, histological scores of hepatic inflammation, and bile duct damage were positively correlated with the degree of splenomegaly. Hepatic CD4+ and CD8+ T cells numbers were significantly higher in mice with splenomegaly, and this was particularly observed in activated effector memory CD4+ T and CD8+ T cells. A proportion of some other immune cells including granulocytes, B, natural killer (NK), and CD8+ T effector memory cells were also altered in the enlarged spleen. More importantly, administration of quadruple antibiotics to deplete gut microbiota relieved the splenomegaly of p40-/-IL-2Rα-/- mice, significantly alleviated liver inflammation, and caused a significant reduction of liver and spleen T cell accumulation and activation; however, single antibiotics did not induce these changes. Conclusions: Splenomegaly was associated with more severe liver inflammation in our PBC murine model, and this effect was reversed by quadruple antibiotic treatment.

The Proinflammatory Cytokines IL-18, IL-21, and IFN-γ Differentially Regulate Liver Inflammation and Anti-Mitochondrial Antibody Level in a Murine Model of Primary Biliary Cholangitis.

Primary biliary cholangitis (PBC) is a cholestatic liver disease primarily featured by autoimmune-mediated damage of intrahepatic small- and medium-sized bile ducts. Elevated serum proinflammatory cytokines, serum anti-mitochondrial antibodies (AMAs), liver inflammation, and fibrosis are also hallmarks of PBC disease. However, whether the elevated proinflammatory cytokines play a role in autoimmune cholangitis remains unknown. Herein, we utilized the p40-/-IL-2Rα -/- PBC mouse model to investigate the roles of proinflammatory cytokines IL-18, IL-21, and IFN-γ in the onset and progression of PBC. IL-18-/-, IFN-γ -/-, and IL-21-/- mice were crossed with p40-/-IL-2Ra+/- mice, respectively, to produce corresponding cytokine-deficient PBC models. Autoantibody level, liver inflammation, and bile duct injury were analyzed. We found that livers from p40-/-IL-2Rα -/- mice exhibit similar transcriptomic characters of PBC patients. In p40-/-IL-2Rα -/- mice, deletion of IL-18 has no remarkable effect on disease progression, while deletion of IL-21 indicates that it is necessary for AMA production but independent of liver inflammation and cholangitis. IFN-γ is responsible for both AMA production and liver inflammation in our model. Our results demonstrate that different proinflammatory cytokines can regulate different effector functions in PBC pathogenesis and need to be considered in PBC treatment.