Knockdown of long noncoding RNA growth arrest-specific transcript 5 regulates forkhead box O3 to inhibit lipopolysaccharide-induced human bronchial epithelial cell pyroptosis.

Pyroptosis is a novel proinflammatory programmed cell death process. This study was designed to investigate the functional mechanisms of long noncoding RNA growth arrest-specific transcript 5 (lncRNA GAS5) on lipopolysaccharide (LPS)-induced human bronchial epithelial cell (HBEC) pyroptosis. LPS was used to induce pyroptosis in HBECs, followed by the detection of the expression of GAS5, forkhead box O3 (FOXO3), and nuclear factor E2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway-related factors. Cell viability was evaluated using CCK-8 assay, lactate dehydrogenase (LDH) release was assessed by LDH assay kit and caspase-1 activity by flow cytometry. Furthermore, expression of NOD-like receptor family pyrin domain containing 3 and pyroptosis-related proteins was evaluated using Western blot analysis, while enzyme-linked immunosorbent assay was used to determine the levels of inflammatory factors. The interaction between GAS5 and FOXO3 was confirmed using bioinformatic prediction, RNA immunoprecipitation assay, RNA pull-down, and dual-luciferase reporter gene assay. Treatment of HBECs with LPS upregulated the expression of GAS5 and FOXO3, resulting in the inactivation of the Nrf2/HO-1 signaling pathway. On the other hand, inhibition of both GAS5 and FOXO3 promoted cell viability, reduced LDH release, pyroptosis, and inflammatory response in LPS-induced HBECs. Furthermore, FOXO3 could interact with GAS5, while FOXO3 overexpression reversed the inhibitory effect of GAS5 knockdown on cell pyroptosis. Thus, mechanistically, inhibition of FOXO3 activates the Nrf2/HO-1 pathway to suppress LPS-induced pyroptosis in HBECs. This study revealed that GAS5 knockdown attenuates FOXO3 expression thereby activating the Nrf2/HO-1 pathway to inhibit LPS-induced pyroptosis in HBECs. These findings may contribute to identifying novel targets that inhibit pyroptosis in HBECs.

SOCS3 Promotes ALV-J Virus Replication via Inhibiting JAK2/STAT3 Phosphorylation During Infection.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that causes immunosuppression and neoplastic diseases in poultry. Cytokine signal-transduction inhibitor molecule 3 (SOCS3) is an important negative regulator of the JAK2/STAT3 signaling pathway and plays certain roles in ALV-J infection. It is of significance to confirm the roles of SOCS3 in ALV-J infection and study how this gene affects ALV-J infection. In this study, we assessed the expression of the SOCS3 gene in vivo and in vitro, and investigated the roles of SOCS3 in ALV-J infection using overexpressed or interfered assays with the SOCS3 in DF-1 cells. The results showed that the SOCS3 expression of ALV-J infected chickens was different from uninfected chickens in the spleen, thymus and cecal tonsil. Further, SOCS3 is mainly expressed in the nucleus as determined by immunofluorescence assay. Overexpression of SOCS3 in DF-1 cells promoted the replication of ALV-J virus, and the expression of interferons (IFNα and INFß), inflammatory factors (IL-6 and TNFα) along with interferon-stimulating genes (CH25H, MX1, OASL, and ZAP). Conversely, interference of SOCS3 showed the opposite results. We also observed that SOCS3 promoted ALV-J virus replication by inhibiting JAK2/STAT3 phosphorylation. In conclusion, SOCS3 promotes ALV-J replication via inhibiting the phosphorylation of the JAK2/STAT3 signaling pathway. These results would advance further understanding of the persistent infection and the viral immune evasion of the ALV-J virus.

Prolactin affects the disappearance of ALV-J viremia in vivo and inhibits viral infection.

Based on the RNA-seq data of chicken spleen tissues infected with J subgroup avian leukosis virus (ALV-J), we found that prolactin (PRL) gene was one of differentially expressed gene. We measured ALV-J viremia and PRL levels in the plasma of two groups of ALV-J-infected adult chickens. Furthermore, recombinant chicken PRL (cPRL) was used to assess how cPRL affects ALV-J virus replication both in vivo and in vitro. The results showed that PRL levels in the plasma of adult chickens infected with ALV-J were lower than those of uninfected chickens, and that the difference was more significant in the avian leukemia pathological apparent changes. Notably, the fluctuations in PRL levels might influence the disappearance of ALV-J viremia in chickens. The in vitro results showed that preincubating DF-1 cells with cPRL before ALV-J infection elicited the best antiviral effects. Moreover, these effects were not dose-dependent. in vivo, injection of cPRL into ALV-J-infected chicks could reduce the levels of viremia at the 14 days post infection (dpi). Additionally, the expression of the interferon-stimulated genes oligoadenylate synthetase-like (OSAL) and vasoactive intestinal peptide (VIP) increased, and that of the proinflammatory cytokine-encoding TNTα, IL-1ß, and IL-6 genes decreased in the spleens of ALV-J-infected chicks injected with cPRL, leading to inhibition of viral replication at the 7 dpi. Collectively, our data demonstrated that PRL plays an important antiviral role in the immune response to ALV-J infection. This is the first report of the relationship between ALV-J infection and PRL. It is of great significance for the prevention and control of ALV-J.

Phylogenetic Analysis of ALV-J Associated with Immune Responses in Yellow Chicken Flocks in South China.

The aim of this study was to better understand the sequence characteristics and immune responses in avian leukosis virus subgroup J (ALV-J) infected yellow chicken flocks in South China. We isolated four strains of ALV-J virus from these flocks, which were then identified by several methods, including subtype-specific polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay (IFA). All four viruses were sequenced for their complete genomes and named GD19GZ01, GD19GZ02, GD19GZ03, and GD19GZ04. In comparison with the reference sequence, the homology analysis showed that the gag and pol genes were relatively conserved, whereas env contained much variation. Both GD19GZ01 and GD19GZ02 almost entirely lacked the rTM region and E element, while the latter was retained in GD19GZ03 and GD19GZ04. Moreover, the virus replication levels in GD19GZ03 and GD19GZ04were much higher than those in GD19GZ01 and GD19GZ02. And three virus recombination events in GD19GZ01 and GD19GZ02 were revealed by the results of PDR5 and SimPlot software analysis. Additionally, we found that some interferon-stimulating genes (CH25H, MX, PKR, OAS, and ZAP) and inflammatory mediators (IL-4, IL-6, IL-10, IL-12, 1L-18, and TNF-α) were significantly upregulated in the immune system organs of clinical chickens. Taken together, these findings clarify and reveal the sequence characteristics and trends in the variation of ALV-J infection in yellow chicken flocks of South China.