HTRA3 Is a Prognostic Biomarker and Associated With Immune Infiltrates in Gastric Cancer.

HtrA serine peptidase 3 (HTRA3) participates in multiple signal pathways and plays an important regulatory role in various malignancies; however, its role on prognosis and immune infiltrates in gastric cancer (GC) remains unclear. The study investigated HTRA3 expression in tumor tissues and its association with immune infiltrates, and determined its prognostic roles in GC patients. Patients with GC were collected from the cancer genome atlas (TCGA). We compared the expression of HTRA3 in GC and normal gastric mucosa tissues with Wilcoxon rank sum test. And logistic regression was used to evaluate the relationship between HTRA3 and clinicopathological characters. Gene ontology (GO) term analysis, Gene set enrichment analysis (GSEA), and single-sample Gene Set Enrichment Analysis (ssGSEA) was conducted to explain the enrichmental pathways and functions and quantify the extent of immune cells infiltration for HTRA3. Kaplan-Meier analysis and Cox regression were performed to evaluate the correlation between HTRA3 and survival rates. A nomogram, based on Cox multivariate analysis, was used to predict the impact of HTRA3 on prognosis. High HTRA3 expression was significantly correlated with tumor histological type, histological grade, clinical stage, T stage, and TP53 status (P < 0.05). HTRA3-high GC patients had a lower 10-year progression-free interval [PFI; hazard ratio (HR): 1.46; 95% confidence interval (CI): 1.02-2.08; P = 0.038], disease-specific survival (DSS; HR: 1.65; CI: 1.08-2.52; P = 0.021) and overall survival (OS; HR: 1.59; CI: 1.14-2.22; P = 0.006). Multivariate survival analysis showed that HTRA3 was an independent prognostic marker for PFI (HR: 1.456; CI: 1.021-2.078; P = 0.038), DSS (HR: 1.650; CI: 1.079-2.522; P = 0.021) and OS [hazard ratio (HR): 1.590; 95% confidence interval (CI):1.140-2.219; P = 0.006]. The C-indexes and calibration plots of the nomogram based on multivariate analysis indicated an effective predictive performance for GC patients. GSEA showed that High HTRA3 expression may activate NF-κB pathway, YAP1/WWTR1/TAZ pathway, and TGFß pathway. There was a negative correlation between the HTRA3 expression and the abundances of adaptive immunocytes (T helper cell 17 cells) and a positive correlation with abundances of innate immunocytes (natural killer cells, macrophages etc.). HTRA3 plays a vital role in GC progression and prognosis and could be a moderate biomarker for prediction for survival after gastrectomy.

Decreased CD4+CD25+CD127dim/- Regulatory T Cells and T Helper 17 Cell Responsiveness to Toll-Like Receptor 2 in Chronic Hepatitis C Patients with Daclatasvir Plus Asunaprevir Therapy.

Direct-acting antivirals (DAAs) not only rapidly inhibited hepatitis C virus (HCV) replication but also modulated innate and adaptive immune response in chronic hepatitis C patients. However, the regulatory activity of DAAs to Toll-like receptor 2 (TLR2) stimulation on CD4+CD25+CD127dim/- regulatory T cells (Tregs) and T helper (Th) 17 cells was not completely understood. In the present study, a total of 23 patients with chronic HCV genotype 1b infection were enrolled, and blood samples were collected at baseline (treatment naive), end of therapy (EOT), and 12 weeks after EOT (SVR12) with daclatasvir plus asunaprevir therapy. TLR2 expression on Tregs and Th17 cells was measured by flow cytometry. Cellular proliferation, cytokine production, and suppressive activity were also tested in purified CD4+CD25+CD127dim/- Tregs in response to the stimulation of Pam3Csk4, an agonist of TLR2. Inhibition of HCV RNA by daclatasvir and asunaprevir did not affect either percentage of Tregs/Th17 cells or TLR2 expression on Tregs/Th17 cells. Pam3Csk4 stimulation also did not influence either cellular proliferation or Tregs/Th17 proportion at each time point. Stimulation with Pam3Csk4 only enhanced the suppressive function and interleukin (IL)-35 production by Tregs purified from baseline, but not those from EOT or SVR12. Similarly, Pam3Csk4 stimulation only elevated Th17 cell frequency of CD4+ T cells from baseline, but not those from EOT or SVR12. Moreover, daclatasvir and asunaprevir therapy did not promote TLR2-induced shift of Tregs toward Th17-like phenotype and function. These data suggested that daclatasvir plus asunaprevir therapy resulted in the decreased responsiveness of Tregs/Th17 cells to TLR2 stimulation in chronic hepatitis C patients, which might provide a novel mechanism underlying DAA-induced immunoregulation.

Interleukin-7 Regulates T Follicular Helper Cell Function in Patients with Chronic Hepatitis C.

Signaling through interleukin (IL)-7 is essential and required for development, differentiation, proliferation, and homeostasis of T cells. However, the role of IL-7 in regulation of CD4+ T cells in chronic viral infections was not fully elucidated. Thus, the aim of the current study was to investigate the immunomodulatory activity of IL-7 to T follicular helper (Tfh) cells and its contribution to pathogenesis of chronic HCV, hepatitis C virus (HCV) infection. A total of 47 patients with chronic hepatitis C and 19 normal controls were enrolled. Serum IL-7 and proportion of Tfh cells was measured. The regulatory function of IL-7 to Tfh cells was also investigated in CD4+ T cells and CD4+ T/HCVcc-infected Huh7.5 cell cocultured system. Serum IL-7 concentration was significantly downregulated in patients with chronic hepatitis C, and was negatively correlated with HCV RNA level. Tfh frequency and Tfh-associated cytokines (IL-21 and IL-6) were also reduced in chronic HCV-infected patients. Moreover, recombinant IL-7 stimulation elevated proportion of Tfh cells and IL-21/IL-6 secretion in both HCV-specific and nonspecific manners. Furthermore, IL-7-treated CD4+ T cells exhibited elevated antiviral activities without killing infected hepatocytes, which presented as inhibition of HCV RNA, induction of antiviral proteins, and promotion of cytokine production (especially IL-21) in cocultured system. This process might be dependent on IL-6 secretion. The current data revealed that IL-7 regulated HCV-specific and nonspecific activated Tfh cells, which might contribute to viral clearance. IL-7 could be a potential therapeutic agent for the treatment of chronic hepatitis C.

Cathepsin F Knockdown Induces Proliferation and Inhibits Apoptosis in Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common cancers in the world. The cathepsin F (CTSF) gene has recently been found to participate in the progression of several types of cancer. However, the clinical characteristics and function of CTSF in GC as well as its molecular mechanisms are not clear. Six GC cell lines and 44 paired adjacent noncancerous and GC tissue samples were used to assess CTSF expression by quantitative polymerase chain reaction (qPCR). We used lentivirus-mediated small hairpin RNA (Lenti-shRNA) against CTSF to knock down the expression of CTSF in GC cells. Western blot and qPCR were used to analyze the mRNA and related protein expression. The biological phenotypes of gastric cells were examined by cell proliferation and apoptosis assays. Microarray-based mRNA expression profile screening was also performed to evaluate the potential molecular pathways in which CTSF may be involved. The CTSF mRNA level was associated with tumor differentiation, depth of tumor invasion, and lymph node metastasis. Downregulation of CTSF expression efficiently inhibited apoptosis and promoted the proliferation of GC cells. Moreover, a total of 1,117 upregulated mRNAs and 1,143 downregulated mRNAs were identified as differentially expressed genes (DEGs). Further analysis identified the involvement of these mRNAs in cancer-related pathways and various other biological processes. Nine DEGs in cancer-related pathways and three downstream genes in the apoptosis pathway were validated by Western blot, which was mainly in agreement with the microarray data. To our knowledge, this is the first report investigating the effect of CTSF on the growth and apoptosis in GC cells and its clinical significance. The CTSF gene may function as a tumor suppressor in GC and may be a potential therapeutic target in the treatment of GC.

Notch Signaling Regulates Circulating T Helper 22 Cells in Patients with Chronic Hepatitis C.

Notch signaling enhanced the response of interleukin (IL)-22-producing CD4+ T cells that were defined as T helper 22 (Th22) cells, and Notch-aryl hydrocarbon receptor (AhR)-IL-22 axis fine-tuned inflammatory response. Previous studies have demonstrated that both Notch signaling and Th22 cells took part in the pathogenesis of chronic hepatitis C virus (HCV) infection. Thus, in this study, we aimed at examining the regulatory role of Notch signaling in Th22 cells in HCV infection. A total of 59 patients with chronic hepatitis C and 22 normal controls (NCs) were enrolled in this study. The percentage of Th22 cells and mRNA expression of related transcriptional factors and cytokines were analyzed in response to γ-secretase inhibitor. Th22 cell frequency was significantly elevated in chronic hepatitis C in comparison with that in NCs. Inhibition of Notch signaling downregulated HCV-specific Th22 cells and IL-22 production, which was accompanied by the reduction of AhR and modulatory cytokines (IL-6 and tumor necrosis factor-α). Moreover, the suppression of Notch signaling also decreased the IL-22-mediated antimicrobial response in both normal and HCV-infected HepG2 cells/Huh7.5 cells. This process was also accompanied by the depression of signal transducers and activators of transcription 3 signaling. In conclusion, the current results suggested that Notch signaling acted as a critical pathway in determining the response to IL-22 in chronic hepatitis C. Thus, Notch-Th22 axis might be considered a new therapeutic target for HCV-infected patients.

Toll-Like Receptor 2 Modulates the Balance of Regulatory T Cells and T Helper 17 Cells in Chronic Hepatitis C.

Regulatory T cells (Tregs) and interleukin-17-producing T helper (Th17) cells were mutually antagonistic in the pathogenesis of chronic hepatitis C virus (HCV) infection. However, the regulation of imbalance between Tregs and Th17 cells was poorly understood in HCV infection. A recent report revealed the immunomodulatory role of Toll-like receptor (TLR) 2 in regulating the balance of Tregs/Th17 functions in multiple sclerosis. Thus, the aim of the current study was to assess the effect of TLR2 stimulation on the suppressive function of Tregs and Th17 differentiation in chronic hepatitis C. A total of 65 patients with chronic hepatitis C receiving pegylated interferon-a2a and ribavirin therapy for 48 weeks, as well as 20 of normal controls (NCs) were enrolled. Cellular proliferation and cytokine production was tested in purified CD4(+)CD25(+)CD127(dim/-) Tregs in response to the stimulation of Pam3Csk4, an agonist of TLR2. In treatment-naive patients, Tregs, but not Th17 cells, from chronic hepatitis C patients expressed higher levels of TLR2 compared with NCs. Stimulation with Pam3Csk4 enhanced the suppressive function of Tregs and production of IL-10 in chronic hepatitis C more than in NCs. However, TLR2 stimulation did not promote Th17 differentiation of Tregs in chronic hepatitis C patients. Moreover, effective anti-HCV therapy resulted in the induction of IL-17-secreting phenotypic shift of Tregs without loss of inhibitive function upon TLR2 stimulation. These data provided a novel mechanism underlying modulating the balance of Tregs/Th17 cells in chronic hepatitis C. HCV infection shifted Tregs/Th17 cells through TLR2 stimulation by inducing Tregs to produce IL-10 and enhancing inhibitive function of effector T cells, resulting in viral persistence.