Blockage of CacyBP inhibits macrophage recruitment and improves anti-PD-1 therapy in hepatocellular carcinoma.

BACKGROUND: Despite remarkable advancements in cancer immunotherapy, the overall response rate to anti-programmed cell death-1 (anti-PD-1) therapy in hepatocellular carcinoma (HCC) patients remains low. Our previous study has demonstrated the critical role of CacyBP/SIP (Calcyclin-Binding Protein and Siah-1 Interacting Protein) as a regulator of HCC development and progression. However, the possible impact of CacyBP on the tumor immune microenvironment has not yet been clarified. METHODS: The expressions of CacyBP and Myd88 in HCC cell lines and tissues was detected by bioinformatics analysis, real-time quantitative PCR, western blotting and immunohistochemistry. The interaction between CacyBP and Myd88 was measured using co-immunoprecipitation and immunofluorescence. In vitro and in vivo assays were used to investigate the regulation of CacyBP on tumor-associated macrophages (TAMs). RESULTS: We identified that CacyBP was positively correlated with Myd88, a master regulator of innate immunity, and Myd88 was a novel binding substrate downstream of CacyBP in HCC. Additionally, CacyBP protected Myd88 from Siah-1-mediated proteasome-dependent degradation by competitively binding to its Toll/interleukin-1 receptor (TIR) domain. Inhibition of CacyBP-Myd88 signaling subsequently diminished HDAC1-mediated H3K9ac and H3K27ac modifications on the CX3CL1 promoter and reduced its transcription and secretion in HCC cells. Moreover, by using in vitro and in vivo strategies, we demonstrated that depletion of CacyBP impaired the infiltration of TAMs and the immunosuppressive state of the tumor microenvironment, further sensitizing HCC-bearing anti-PD-1 therapy. CONCLUSIONS: Our findings suggest that targeting CacyBP may be a novel treatment strategy for improving the efficacy of anti-PD-1 immunotherapy in HCC.

Erythroid-transdifferentiated myeloid cells promote portal vein tumor thrombus in hepatocellular carcinoma.

Rationale: Hepatocellular carcinoma (HCC) is primarily characterized by a high incidence of vascular invasion. However, the specific mechanism underlying portal vein tumor thrombus (PVTT) in HCC remains unclear. As a consequence of myeloid cell developmental arrest, CD71+ erythroid progenitor cells (EPCs) and myeloid-derived suppressor cells play important roles in HCC; however, their roles in PVTT remain unclear. Methods: The role of CD71+ EPCs in the HCC tumor microenvironment (TME) was evaluated via morphological, RNA-sequencing, enzyme-linked immunosorbent assay, and flow cytometric analyses. Co-culture techniques were employed to assess the CD45+ EPCs and their vascular compromising effect. Additionally, the PVTT-promoting function of CD45+ EPCs was explored in vivo in a murine model. Results: The CD45+EPCs in HCC tissues exhibited increased myeloid cell features, including morphology, surface markers, transforming growth factor (TGF)-ß generation, and gene expression, compared with those in circulation. Hence, a large proportion of CD45+EPCs, particularly those in TMEs, comprise erythroid-transdifferentiated myeloid cells (EDMCs). Additionally, the expression of C-C chemokine receptor type 2 (CCR2) mRNA was upregulated in CD45+EPCs within the TME. Tumor macrophages from HCC tissues induced substantial migration of CD45+EPCs in a dose-dependent manner. Meanwhile, results from immunofluorescence analyses revealed that these two cell types are positively associated in the TME and circulation. That is, EDMCs are chemoattracted by HCC macrophages mainly via CCR2 from CD45+ EPCs in the circulation. Additionally, the expressions of FX, FVII, FGB, C4b, CFB, and CFH were elevated in CD45+EPCs within the TME compared with those in the spleen. The CD45+EPCs from the HCC TME promoted vessel endothelial cell migration and compromised tube formation through TGF-ß and FGB, respectively. Additionally, CD45+EPCs from the TME induced HCC cell migration. HCC macrophage-induced CD45+EPCs to exhibit higher levels of FX, FVII, FGB, and TGF-ß. Meanwhile, upregulation of CCAAT/enhancer binding protein beta expression induced FGB and TGF-ß generation in CD45+EPCs in the TME. WTAP, a major RNA m6A writer, stabilized FX and FVII mRNA and enhanced their nuclear export in CD45+EPCs from the TME. CD45+EPCs from the TME were positively associated with PVTT and poor prognosis. Splenectomy reduced the level of CD45+EPCs in the circulation and TME, as well as the incidence of microvascular invasion. The incidence of microvascular invasion increased following the transfer of HCC tissue CD45+EPCs to splenectomized HCC-bearing mice. Conclusions: The CD45+EPCs enriched in the HCC microenvironment are EDMCs, which are induced by HCC macrophages to migrate from the circulation to the TME. Subsequently, EDMCs promote PVTT by compromising the blood vessel endothelium, aggravating coagulation, and promoting HCC cell migration.

LAG3+ erythroid progenitor cells inhibit HBsAg seroclearance during finite pegylated interferon treatment through LAG3 and TGF-ß.

HBsAg seroclearance, the ideal aim of anti-hepatitis B virus (HBV) treatment, cannot be achieved easily. Anemia is another common issue for chronic hepatitis B (CHB) patients, which leads to elevation of erythroid progenitor cells (EPCs) and immune suppression in cancer. This study investigated the role of EPCs in HBsAg seroclearance following pegylated interferon-α (PEG-IFN) treatment. CD45+EPC accumulation in CHB patients and an AAV/HBV mice model was found in the circulation and liver by flow cytometry and immunofluorescence tests. Wright-Giemsa staining showed that these pathological CD45+EPCs presented elevated erythroid cells with relative immature morphologies and atypical cells compared with the control cells. CD45+EPCs were associated with immune tolerance and decreased HBsAg seroclearance during finite PEG-IFN treatment. CD45+EPCs suppressed antigen non-specific T cell activation and HBV-specific CD8+T cells, partially through transforming growth factor ß (TGF-ß). RNA-seq revealed that CD45+EPCs in patients with CHB presented a distinct gene expression profile compared with CD45-EPCs and CD45+EPCs from cord blood. Notably, CD45+EPCs from patients with CHB expressed high level of Lymphocyte-activation gene 3 (LAG3), an immune checkpoint molecule, and were then defined as LAG3+EPCs. LAG3+EPCs diminished the function of antigen presenting cells through LAG3, which was another mechanism by which LAG3+EPCs' suppressed HBV-specific CD8+T cells. Anti-LAG3 and anti-TGF-ß combination treatment decreased serum HBeAg, HBV DNA levels and HBsAg level, as well as HBsAg-expression in hepatocytes during PEG-IFN treatment in the AAV/HBV mice model. Conclusions: LAG3+EPCs inhibited the efficacy of PEG-IFN treatment on HBsAg seroclearance induced by LAG3 and TGF-ß. Anti-LAG3, anti-TGF-ß and PEG-IFN combination treatment might facilitate HBV clearance.

COLEC10 Induces Endoplasmic Reticulum Stress by Occupying GRP78 and Inhibits Hepatocellular Carcinoma.

Collectin subfamily member 10 (COLEC10), a C-type lectin mainly expressed in the liver, is involved in the development of hepatocellular carcinoma (HCC). However, its underlying molecular mechanism in HCC progression remains unknown. In this study, reduced COLEC10 expression in tumor tissues was validated using various HCC cohorts and was associated with poor patient prognosis. COLEC10 overexpression attenuated HCC cell growth and migration abilities in vitro and in vivo. We identified that COLEC10 was a novel interactor of 78-kDa glucose-regulated protein (GRP78), a master modulator of the unfolded protein response in the endoplasmic reticulum (ER). COLEC10 overexpression potentiated ER stress in HCC cells, as demonstrated by elevated expression levels of phosphorylated protein kinase RNA-like ER kinase, phosphorylated inositol-requiring protein 1α, activating transcription factor 4, DNA damage-inducible transcript 3, and X-box-binding protein 1s. The ER in COLEC10-overexpressing cells also showed a dilated and fragmented pattern. Mechanistically, COLEC10 overexpression increases GRP78 occupancy through direct binding by the C-terminal carbohydrate recognition domain in the ER, which released and activated the ER stress transducers protein kinase RNA-like ER kinase and phosphorylated inositol-requiring protein 1α, triggering the unfolded protein response activity. COLEC10-overexpressing HCC cells generated a relatively high reactive oxygen species level and switched to apoptotic cell death under sorafenib-treated conditions. Our study provides the first novel view that COLEC10 inhibits HCC progression by regulating GRP78-mediated ER stress signaling and may serve as a promising therapeutic and prognostic biomarker.

Transcriptomic and proteomic analysis of Staphylococcus aureus response to cuminaldehyde stress.

The previous study indicated that cuminaldehyde (CUM) could be used as an antibacterial agent in sauced beef to reduce the propagation of Staphylococcus aureus (S. aureus). This research took sauced beef treated with 0.4 µL/mL CUM as the research object. Transcriptomic and proteomic methods were used to comprehensively analyze the changes in genes and proteins of S. aureus under CUM stress. A total of 258 differentially expressed genes (DEGs, 178 up-regulated and 80 down-regulated) and 384 differentially expressed proteins (DEPs, 61 up-regulated and 323 down-regulated) were found. It was observed that CUM destroyed the cell wall and cell membrane by inhibiting the synthesis of peptidoglycan and fatty acid. Low energy consumption strategies were formed by reducing glycolysis and ribosome de novo synthesis. The levels of genes and proteins associated with the glycine, serine, threonine, methionine, cysteine, and branched-chain amino acids were dramatically changed, which impaired protein synthesis and reduced bacterial viability. In addition, the up-regulated DEGs and DEFs involved in DNA replication, recombination and single-stranded DNA-binding contributed to DNA repair. Moreover, ATP-binding cassettes (ABC) transporters were also perturbed, such as the uptake of betaine and iron were inhibited. Thus, this study revealed the response mechanism of S. aureus under the stress of CUM, and provided a theoretical basis for the application of CUM in meat products.

Comprehensive Analysis of ANLN in Human Tumors: A Prognostic Biomarker Associated with Cancer Immunity.

Background: Anillin (ANLN), a ubiquitously expressed actin-binding protein, plays a critical tumor-promoting role in cell growth, migration, and cytokinesis. Numerous studies have suggested that ANLN is upregulated in many cancer types, as well as significantly associated with patient prognosis and malignant cancer characteristics. Herein, we performed an integrated pan-cancer analysis of ANLN and highlighted its underlying mechanism, which may benefit further exploration of the potential therapeutic options for cancer. Methods: ANLN expression data were extracted from online databases, including TCGA, GTEx, and CCLE databases. The TIMER database was used to study the association between ANLN expression with immune checkpoint genes and immunocyte infiltration. The ScanNeo pipeline was adopted for neoantigen discovery. KEGG analysis and the STRING tool were used to elucidate the potential mechanism of ANLN in cancer development. Results: ANLN is abnormally overexpressed in almost all cancer tissues compared with normal tissues. The high-ANLN expression level was positively associated with various malignant characteristics, suggesting its potential role in the immune microenvironment and poor prognosis. In addition, ANLN expression was correlated with the number of neoantigens and different phosphorylation pattern in various cancer types, revealing a functional role of genetic mutation accumulation and high phosphorylation in ANLN-mediated oncogenesis. Moreover, we found that ANLN was an important regulatory factor participating in many signaling events, especially the cell cycle and nucleocytoplasmic transport pathways. Conclusions: ANLN expression is generally overexpressed in various types of cancers, and it may have an important influence on tumor progression and development. ANLN expression is significantly associated with the immune checkpoint biomarkers and tumor immunity. Together, these findings suggest that ANLN may be a predictive marker for patient prognosis across cancers.

Expression and clinical significance of inhibitory receptor Leukocyte-associated immunoglobulin-like receptor-1 on peripheral blood T cells of chronic hepatitis B patients: A cross-sectional study.

ABSTRACT: Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is an inhibitory receptor that is expressed on the surface of multiple immune cells and plays key roles in immune modulation. In patients with chronic hepatitis B (CHB), T cell number and functions are abnormal and the expression of inhibitory receptors is elevated. However, the expression of LAIR-1 on T cells in patients with CHB is still undetermined.We recruited 320 patients with CHB in different disease phases and 17 healthy donors. Serum biochemical and virological examinations were performed for each participant, and their demographic and clinical data were collected. According to the latest American Association for the Study of Liver Disease guidelines, we categorized the patients into 4 groups: immune active, immune tolerant, inactive CHB, and gray zone. Additionally, we tested the expression of LAIR-1 on T cells and T cell subsets using flow cytometry.We observed a significant decrease in LAIR-1 expression on CD3+ T cells and its two subsets (CD4+ and CD8+ T cells) in patients with CHB. LAIR-1 expression on T cells was the lowest in the immune active group. LAIR-1 expression levels on CD4+ and CD8+ T cells showed a significant negative association with hepatitis B virus (HBV) DNA load and were lower in hepatitis B e antigen (HBeAg)-positive patients than in HBeAg-negative patients (P < .05). In addition, LAIR-1 expression levels on CD3+, CD4+, and CD8+ T cells were all negatively associated with liver inflammation and fibrosis parameters, such as alanine aminotransferase and aspartate aminotransferase levels, FibroScan value, and aspartate aminotransferase-to-platelet ratio index score.LAIR-1 expression levels on T cells were associated with HBV DNA load and liver inflammation and fibrosis parameters, indicating that LAIR-1 may play an important regulatory role in HBV-induced T cell immune pathogenesis and may be a therapeutic target for CHB.

Serum HBV pregenomic RNA exhibited opposite associations with NKdim and NKbright cell immunity in treatment-naïve chronic hepatitis B patients.

Hepatitis B virus (HBV) pregenomic RNA (pgRNA) is a new biomarker that reflects HBV replication, but its relationship with natural killer (NK) cell immunity in chronic hepatitis B (CHB) is unknown. We assessed serum HBV pgRNA levels in 323 CHB patients by reverse transcription-polymerase chain reaction, assessed cytokine production and activation and inhibitory markers of NK cells by flow cytometry, and measured serum cytokines by enzyme-linked immunosorbent assays (ELISAs). Among the different CHB phases, the serum HBV pgRNA level was highest in the immune-tolerant (IT) and immune-active (IA) phases. Regarding NK and NKdim cells, HBV pgRNA was negatively associated with frequencies, but positively associated with NKp44 and NKp46 expression (activation markers). Regarding NKbright cells, serum HBV pgRNA was positively associated with frequency and programmed cell death protein 1 (PD1) expression (inhibitory marker), but negatively associated with NKp44 and NKp46. Serum HBV pgRNA was not associated with NKp30 (activation marker) on NK cells or subsets. Lastly, serum HBV pgRNA was positively correlated with the levels of serum IL-7 and IL-12P40 (NK cell-promoting cytokines) and negatively correlated with serum prostaglandin E2 (PGE2) level (which negatively regulates NK cells). In conclusion, we found varied relationships between serum HBV pgRNA and NK cells and subsets, indicating that HBV pgRNA may play a complicated role in NK cell-related immunity, providing new information on HBV and host immunity.

Association among cytokine profiles of innate and adaptive immune responses and clinical-virological features in untreated patients with chronic hepatitis B.

BACKGROUND: Complete clearance of intracellular viruses depends on effector cells of innate and adaptive immune systems. This study aimed to identify the relationships among antiviral cytokines produced by natural killer (NK) and T cells and clinical-virological characteristics in untreated chronic hepatitis B (CHB) patients. METHODS: We measured antiviral cytokines interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin-2 (IL-2) produced by T, NK and natural killer T (NKT) cells, respectively, in a cohort with chronic hepatitis B virus (HBV) infection (CHB). We also correlated these cytokines with clinical-virological characteristics using a linear regression model. RESULTS: levels of IFN-γ+ and TNF-α+ CD4+ and CD8+ T cells were significantly higher in immune active (IA) phase than in other phases. Immune tolerant (IT) patients showed the lowest expression of IFN-γ by NK and NKT cells, and TNF-α by NK cells. IFN-γ+, TNF-α+ and IL-2+ CD4+ and CD8+ T cells frequencies were similar between IA and gray zone (GZ) phases. Principal component analysis based on cytokines confirmed that most IT patients significantly differed from inactive carriers (IC) and IA patients, while GZ patients were widely scattered. Multivariate analysis showed both T and NK cells producing IFN-γ and TNF-α, but not IL-2, had significant association with serum alanine aminotransferase (ALT). Moreover, IFN-γ+ NKT cells were associated with HBV DNA, while IFN-γ+ CD4+ and CD8+ T cells were correlated with age. CONCLUSION: HBV clinical phases are characterized by distinct cytokine signatures, which showed relationship to viral features in these untreated CHB patients.

CCL14 is a prognostic biomarker and correlates with immune infiltrates in hepatocellular carcinoma.

C-C motif chemokine ligand 14 (CCL14) is a chemokine promoting the activation of immune cells. However, the relationship between CCL14 expression, tumor immunity, and prognosis in Hepatocellular Carcinoma (HCC) remain unclear. CCL14 expression and its influence on tumor prognosis were analyzed by the ONCOMINE, Tumor Immune Estimation Resource (TIMER) and Kaplan-Meier plotter. The relationship between CCL14 expression and tumor immunity were analyzed by TIMER and Gene Expression Profiling Interactive Analysis (GEPIA). CCL14 expression was significantly lower in several human cancers, including HCC, than in corresponding normal tissues. CCL14 expression in HCC tissues correlated with prognosis. Low CCL14 expression associated with poorer overall survival, disease-specific survival, progression-free survival, and relapse-free survival in multiple cohorts of HCC patients, particularly at early disease stages (stage 1+2 or grade 2). CCL14 showed strong correlation with tumor-infiltrating B cells, CD4+ and CD8+ T cells, macrophages, neutrophils, and dendritic cells. CCL14 expression in HCC negatively correlated with expression of several immune cell markers, including exhausted T cell markers, PD-1, TIM-3 and CTLA-4, suggesting its role in regulating tumor immunity. These findings demonstrate that CCL14 is a potential prognostic biomarker that determines cancer progression and correlated with tumor immune cells infiltration in HCC.

An Immuno-Clinic score model for evaluating T cell immunity and predicting early antiviral therapy effectiveness in chronic hepatitis B.

We generated an Immuno-Clinic score (ICS) model to evaluate T cell immunity based on the clustering of antiviral cytokines and inhibitory molecules in 229 naïve chronic hepatitis B (CHB) patients. 126 patients receiving antiviral therapy were used to validate the model for predicting antiviral therapy effectiveness. Through receiver-operator characteristic curve analysis, the area under the curve, sensitivity, and specificity of the ICS model were 0.801 (95%CI 0.703-0.900), 0.727, and 0.722, respectively. The cut-off value was 0.442. Re-evaluation of T cell immunity in different phases of CHB showed that patients in the immune tolerant phase had the lowest percentage of ICS-high (15%), while patients in the inactive carrier phase had the highest percentage of ICS-high (92%). Patients in the immune active and gray zone phases had 17% and 56% ICS-high, respectively. Elevation of ICS as early as four weeks after treatment could predict the effectiveness of hepatitis B virus (HBV) DNA loss and normalization of alanine aminotransferase, while eight weeks after treatment could predict HBV surface antigen decline. Thus, this ICS model helps clinicians choose an optimal time for initiating antiviral therapy and predicting its efficacy.

Serum HBV pregenomic RNA is correlated with Th1/Th2 immunity in treatment-naïve chronic hepatitis B patients.

BACKGROUND AND AIM: Hepatitis B virus (HBV) load and antigens are related to the innate and adaptive immunity of chronic hepatitis B (CHB) patients. As a new HBV biomarker, the role of pregenomic RNA (pgRNA) in host immunity is not known. This study aimed to identify the relationship between serum HBV pgRNA and host immunity in CHB patients. METHODS: Two hundred twenty-five treatment-naïve CHB patients were enrolled. Serum cytokines were measured by cytokine antibody array (Luminex multiplex platform). Th1 (T-helper cell, Th) and Th2 cells were tested by flow cytometry. Serum HBV pgRNA was detected by a reverse transcription-polymerase chain reaction. RESULTS: Serum HBV pgRNA was significantly different among patients in different disease phases and significantly associated with both HBV antigens and antibodies. Serum HBV pgRNA was positively correlated with the HBsAg level (P < .001) and the presence of HBeAg (P < .001). Patients with higher HBcAb levels showed lower serum HBV pgRNA levels (P = .003). Notably, HBsAb positivity was associated with higher levels of serum HBV pgRNA in HBeAg(-) patients (P = .049). Serum HBV pgRNA was positively associated with ALT level, Th2 cell frequency, and related cytokine sCD30 (P < .001, P < .001, and P = .003, respectively), but negatively associated with Th1-related cytokine interleukin (IL)-12P70 and cytotoxic lymphocytes (CTLs) (P = .017 and P < .001, respectively). CONCLUSION: Our study confirmed the relationship between serum HBV pgRNA and host immunity. The results demonstrated that serum HBV pgRNA is positively correlated with Th2 immunity but negatively correlated with Th1 immunity, indicating that it might have a relationship with HBV antigen conversion and CTL immunodeficiency in CHB patients.