CX3CR1+ macrophages interact with hepatic stellate cells to promote hepatocellular carcinoma through CD8+ T cell suppression.

BACKGROUND AND AIMS: Hepatic stellate cells (HSCs) contribute to hepatocellular carcinoma (HCC) progression by regulating multiple factors. However, the entire immunoregulatory functions of HSCs are still obscure. Here, we aim to investigate whether HSCs impose CX3CR1+ macrophages to pro-tumorigenic properties in the peritumoral area. APPROACH AND RESULTS: In single cell RNA-sequencing analysis of HCC patients, a subpopulation of macrophages specifically expressed Arg1 and Cx3cr1 in the peritumoral area, and were highly enriched with retinol metabolism-related genes. Flow cytometry analysis showed significantly increased frequencies of CD14+CD11b+HLA-DR‒ macrophages with CX3CR1 in the HCC adjacent region where α-SMA-expressing activated HSCs (aHSCs) showed co-localized expression of CX3CL1. Accordingly, in tumor-bearing mice, Cx3cl1 mRNA expression was notably increased in aHSCs within the adjacent HCC, where infiltration of CX3CR1+Ly6C+ macrophages was mostly observed with decreased CD8+ T cells. In adoptive transfer and in vitro co-culture of myeloid cells, we demonstrated that CX3CR1+Ly6C+ macrophages migrated and highly expressed arginase-1 by interacting with retinoid-enriched aHSCs in the adjacent HCC. Direct treatment of retinoids or co-culturing with retinol-storing mouse aHSCs or human LX-2 cells significantly increased arginase-1 expression in CX3CR1+Ly6C+ macrophages and human blood CD14+ cells, leading to the suppression of CD8+ T cell proliferation. Moreover, genetic deficiency of CX3CR1 in myeloid cells or pharmacological inhibition of retinol metabolism remarkably attenuated HCC development. CONCLUSION: We showed that CX3CR1+Ly6C+ macrophages migrate and interact with aHSCs in the peritumoral region where retinoids induce arginase-1 expression in CX3CR1+Ly6C+ macrophages, subsequently depriving CD8+ T cells of arginine and promoting HCC.

The role of glial and neuronal Eph/ephrin signaling in Drosophila mushroom body development and sleep and circadian behavior.

The Eph receptor, a prototypically large receptor protein tyrosine kinase, interacts with ephrin ligands, forming a bidirectional signaling system that impacts diverse brain functions. Eph receptors and ephrins mediate forward and reverse signaling, affecting neurogenesis, axon guidance, and synaptic signaling. While mammalian studies have emphasized their roles in neurogenesis and synaptic plasticity, the Drosophila counterparts are less studied, especially in glial cells, despite structural similarities. Using RNAi to modulate Eph/ephrin expression in Drosophila neurons and glia, we studied their roles in brain development and sleep and circadian behavior. Knockdown of neuronal ephrin disrupted mushroom body development, while glial knockdown had minimal impact. Surprisingly, disrupting ephrin in neurons or glial cells altered sleep and circadian rhythms, indicating a direct involvement in these behaviors independent from developmental effects. Further analysis revealed distinct sleep phenotypes between neuronal and glial knockdowns, underscoring the intricate interplay within the neural circuits that govern behavior. Glia-specific knockdowns showed altered sleep patterns and reduced circadian rhythmicity, suggesting an intricate role of glia in sleep regulation. Our findings challenge simplistic models of Eph/ephrin signaling limited to neuron-glia communication and emphasize the complexity of the regulatory networks modulating behavior. Future investigations targeting specific glial subtypes will enhance our understanding of Eph/ephrin signaling's role in sleep regulation across species.

First detection and characterization of hepatitis E virus (Rocahepevirus ratti) from urban Norway rats (Rattus norvegicus) in the Republic of Korea.

Hepatitis E virus (HEV), an emerging zoonotic pathogen, poses a significant public health concern worldwide. Recently, rat HEV (Rocahepevirus ratti genotype C1; HEV-C1) has been reported to cause zoonotic infections and hepatitis in humans. Human infections with HEV-C1 are considered to be underestimated worldwide due to limited knowledge of transmission routes, genome epidemiology, and the risk assessment of zoonosis associated with these viruses. A total of 186 wild Norway rats (Rattus norvegicus) were collected from the Republic of Korea (ROK) between 2011 and 2021. The prevalence of HEV-C1 RNA was 8 of 180 (4.4%) by reverse-transcription polymerase chain reaction. We first reported three nearly whole-genome sequences of HEV-C1 newly acquired from urban rats in the ROK. Phylogenetic analysis demonstrated that Korea-indigenous HEV-C1 formed an independent genetic group with those derived from R. norvegicus rats in other countries, indicating geographical and genetic diversity. Our findings provide critical insights into the molecular prevalence, genome epidemiology, and zoonotic potential of Rocahepevirus. This report raises awareness of the presence of Rocahepevirus-related hepatitis E among physicians in the ROK.

Aortic carboxypeptidase-like protein, a putative myokine, stimulates the differentiation and survival of bone-forming osteoblasts.

A new target that stimulates bone formation is needed to overcome limitations of current anti-osteoporotic drugs. Myokines, factors secreted from muscles, may modulate it. In this study, we investigated the role of aortic carboxypeptidase-like protein (ACLP), which is highly expressed in skeletal muscles, on bone formation. MC3T3-E1 cells and/or calvaria osteoblasts were treated with recombinant N-terminal mouse ACLP containing a signal peptide [rmACLP (N)]. The expression and secretion of ACLP were higher in skeletal muscle and differentiated myotube than in other tissues and undifferentiated myoblasts, respectively. rmACLP (N) increased bone formation, ALP activity, and phosphorylated p38 mitogen-activated protein (MAP) kinase in osteoblasts; reversal was achieved by pre-treatment with a TGF-ß receptor inhibitor. Under H2 O2 treatment, rmACLP (N) increased osteoblast survival, phosphorylated p38 MAP kinase, and the nuclear translocation of FoxO3a in osteoblasts. H2 O2 treatment caused rmACLP (N) to suppress its apoptotic, oxidative, and caspase-9 activities. rmACLP (N)-stimulated osteoblast survival was reversed by pre-treatment with a p38 inhibitor, a TGF-ß-receptor II blocking antibody, and a FoxO3a shRNA. Conditioned media (CM) from muscle cells stimulated osteoblast survival under H2 O2 treatment, in contrast to CM from ACLP knockdown muscle cells. rmACLP (N) increased the expressions of FoxO3a target anti-oxidant genes such as Sod2, Trx2, and Prx5. In conclusion, ACLP stimulated the differentiation and survival of osteoblasts. This led to the stimulation of bone formation by the activation of p38 MAP kinase and/or FoxO3a via TGF-ß receptors. These findings suggest a novel role for ACLP in bone metabolism as a putative myokine.

Rapid interrogation of cancer cell of origin through CRISPR editing.

The increasing complexity of different cell types revealed by single-cell analysis of tissues presents challenges in efficiently elucidating their functions. Here we show, using prostate as a model tissue, that primary organoids and freshly isolated epithelial cells can be CRISPR edited ex vivo using Cas9-sgRNA (guide RNA) ribotnucleoprotein complex technology, then orthotopically transferred in vivo into immunocompetent or immunodeficient mice to generate cancer models with phenotypes resembling those seen in traditional genetically engineered mouse models. Large intrachromosomal (∼2 Mb) or multigenic deletions can be engineered efficiently without the need for selection, including in isolated subpopulations to address cell-of-origin questions.

Prognosis Following Sustained Virologic Response in Korean Chronic Hepatitis C Patients Treated with Sofosbuvir-Based Treatment: Data from a Multicenter Prospective Observational Study up to 7 Years.

Background and Objectives: Chronic hepatitis C (CHC) can be cured with direct-acting antiviral (DAA) therapy. In Korea, sofosbuvir (SOF) and ledipasvir (LDV)/SOF were launched in 2016. Patients who achieve a sustained virologic response (SVR) following DAA treatment are predicted to have a favorable prognosis. Nevertheless, little is known regarding the prognosis of Korean CHC patients who receive SOF-based treatment and achieve SVR. Therefore, the purpose of this study was to look into the long-term outcomes for these patients. Materials and Methods: This was a prospective, multicenter observational study. CHC patients were enrolled who, following SOF or LDV/SOF treatment, had achieved SVR. The last day for follow-up was December 2023. The primary endpoint was HCC occurrence, which was checked at least once per year. Results: A total of 516 patients were included in this analysis, with a median follow-up duration of 39.0 months. Among them, 231 were male patients (44.8%), with a median age of 62.0 years. Genotypes were 1 (90, 17.4%), 2 (423, 82.0%), and 3 (3, 0.6%). The combination of SOF plus ribavirin was the most common treatment (394, 76.4%). In total, 160 patients were cirrhotic (31.0%), and the mean Child-Pugh score was 5.1. Within a maximum of 7 years, 21 patients (4.1%) developed HCC. Patients with HCC were older (69 vs. 61 years, p = 0.013) and had a higher cirrhosis incidence (81.0 vs. 28.9%, p < 0.001), higher AFP (6.0 vs. 3.3, p = 0.003) and higher APRI (0.8 vs. 0.5, p = 0.005). Age over 65 (p = 0.016) and cirrhosis (p = 0.005) were found to be significant risk factors for HCC by Cox regression analysis. Conclusions: Patients who achieved SVR with SOF-based treatment had a relatively favorable prognosis. However, the risk of HCC was not eliminated, especially in older and cirrhotic patients. Therefore, routine follow-up, surveillance, and early treatment are required.

The clinical effect of antiviral therapy in patients with hepatitis B virus-related decompensated cirrhosis and undetectable DNA.

BACKGROUND AND AIM: Antiviral therapy (AVT) is the mainstay of hepatitis B virus (HBV) management. We investigated whether AVT improves the outcomes of HBV-related decompensated cirrhosis and undetectable HBV-DNA. METHODS: Between 2000 and 2017, treatment-naïve patients with HBV-related decompensated cirrhosis and undetectable HBV-DNA were recruited from two tertiary hospitals. The endpoints included death and hepatocellular carcinoma (HCC). RESULTS: A total of 429 patients were analyzed (50 and 379 patients in the AVT and non-AVT groups, respectively). Patients in the AVT group were significantly younger and had higher alanine aminotransferase and alpha-fetoprotein levels than those in the non-AVT group (all P < 0.05). During follow-up (median 49.6 months), 98 patients died and 105 developed HCC. The cumulative incidence rates of death (2.0%, 4.1%, and 6.4%, and 4.9%, 7.2%, and 10.2% at 6 months, 1 year, and 2 years, respectively) and HCC (8.6%, 15.8%, and 26.4% vs 1.6%, 7.7%, and 24.4% at 1, 2, and 5 years, respectively) were statistically comparable between the AVT and non-AVT groups (all P > 0.05). Using Cox regression analysis, AVT was not significantly associated with death nor HCC (all P > 0.05). Similar results were observed after balancing baseline characteristics with inverse probability of treatment weighting. In the non-AVT group, the cumulative incidence rates of HBV-DNA detection at 6 months, 1 year, and 2 years were 2.0%, 3.1%, and 6.4%, respectively. CONCLUSIONS: Antiviral therapy did not attenuate the risk of death nor HCC in patients with HBV-related decompensated cirrhosis and undetectable HBV-DNA.

Prognosis of Patients with Chronic Hepatitis C Genotype 1b Infection Treated Using Daclatasvir/Asunaprevir after Sustained Virologic Response: A 6-Year Multicenter Prospective Observational Study.

Aim and Objectives: Direct-acting antiviral (DAA) therapy can cure chronic hepatitis C (CHC), and daclatasvir (DCV)/asunaprevir (ASV) was the first interferon-free DAA therapy introduced in Korea. Patients who achieve sustained virologic response (SVR) after DAA treatment are expected to have good prognoses. Therefore, in this study, we aimed to investigate the prognosis of these patients. Materials and Methods: This multicenter prospective observational study included patients with CHC who achieved SVR after DCV/ASV treatment. The primary endpoint was hepatocellular carcinoma (HCC) occurrence, which was reviewed annually. Results: We included 302 patients (median follow-up duration: 38 [16.5-60.0] months; median age: 58 [49-67] years) in the study. Cirrhosis was observed in 103 patients (34.1%), and the median Child-Pugh score was 5.0. HCC occurred in 16 patients (5.3%) within six years post-SVR; these patients were older and had higher cirrhosis prevalence, alpha-fetoprotein levels, and fibrosis-4 index scores than did those without HCC development. Cox proportional hazards analysis revealed that age > 71 years (p = 0.005) and cirrhosis (p = 0.035) were significant risk factors for HCC occurrence. Conclusions: Although the prognoses of patients who achieved SVR with DCV/ASV therapy were generally good, the risk for HCC was present, especially in older patients and in those with cirrhosis. Hence, early treatment at younger ages and regular follow-up surveillance after achieving SVR are warranted.

DNA Methylation Patterns According to Fatty Liver Index and Longitudinal Changes from the Korean Genome and Epidemiology Study (KoGES).

The role of differentially methylated regions (DMRs) in nonalcoholic fatty liver disease (NAFLD) is unclear. This study aimed to identify the role of DMR in NAFLD development and progression using the Korean Genome and Epidemiology Study (KoGES) cohort. We used laboratory evaluations and Illumina Methylation 450 k DNA methylation microarray data from KoGES. The correlation between fatty liver index (FLI) and genomic CpG sites was analyzed in 322 subjects. Longitudinal changes over 8 years were confirmed in 33 subjects. To identify CpG sites and genes related to FLI, we obtained enrichment terms for 6765 genes. DMRs were identified for both high (n = 128) and low (n = 194) groups on the basis of FLI 30 in 142 men and 180 women. To confirm longitudinal changes in 33 subjects, the ratio of follow-up and baseline investigation values was obtained. Correlations and group comparisons were performed for the 8 year change values. PITPNM3, RXFP3, and THRB were hypermethylated in the increased FLI groups, whereas SLC9A2 and FOXI3 were hypermethylated in the decreased FLI groups. DMRs describing NAFLD were determined, and functions related to inflammation were identified. Factors related to longitudinal changes are suggested, and blood circulation-related functions appear to be important in the management of NAFLD.

Associations Between Plasma Growth and Differentiation Factor-15 with Aging Phenotypes in Muscle, Adipose Tissue, and Bone.

Growth and differentiation factor 15 (GDF-15) is associated with muscle, fat, and bone metabolism; however, this association has not been well characterized. Plasma GDF-15, appendicular skeletal muscle mass (ASM), fat mass (FM), and bone mineral density (BMD) were measured in 146 postmenopausal women. GDF-15 levels were higher in subjects with low Body Mass Index (BMI)-adjusted ASM than in those without (median [interquartile range] 831.3 [635.4-1011.4] vs. 583.8 [455.8-771.1] pg/mL, p = 0.018). The GDF-15 level was inversely correlated with BMI-adjusted ASM (r = - 0.377, p < 0.001) and BMD at femur neck (FN-BMD; r = - 0.201, p = 0.015), and positively correlated with percent FM (pFM; r = 0.328, p < 0.001). After adjusting for confounders, the GDF-15 level was inversely associated with BMI-adjusted ASM (ß = -0.250, p = 0.006) and positively associated with pFM (ß = 0.272, p = 0.004), and tended to be inversely associated with FN-BMD (ß = - 0.176, p = 0.076). The area under the receiver-operating characteristic curve of GDF-15 level > 618.4 pg/mL for sarcopenia was 0.706 (95% confidence interval (CI) 0.625-0.779) with a sensitivity of 83.3% and a specificity of 54.5%. Using a GDF-15 level of 618.4 pg/mL as a cut-off, the GDF-15 level was associated with a significantly greater likelihood of sarcopenia (odds ratio [OR] 2.35; 95% CI 1.00-5.51; p = 0.049), obesity (OR 3.28; 95% CI 1.48-7.27; p = 0.001), osteopenic obesity (OR 3.10; 95% CI 1.31-7.30; p = 0.010), and sarcopenic or osteosarcopenic obesity (OR 4.84; 95% CI 0.88-26.69; p = 0.070). These findings support the potential of GDF-15 as a biomarker for age-related changes in muscle, fat, and bone.