Amplification of immunity by engineering chicken MDA5 combined with the C terminal domain (CTD) of RIG-I.

Innate immune system is triggered by pattern recognition receptors (PRRs) recognition. Retinoic acid-inducible gene 1 (RIG-I) is a major sensor that recognizes RNA ligands. However, chickens have no homologue of RIG-I; instead, they rely on melanoma differentiation-associated protein 5 (MDA5) to recognize RNA ligands, which renders chickens susceptible to infection by influenza A viruses (IAVs). Here, we engineered the cMDA5 viral RNA sensing domain (C-terminal domain, CTD) such that it functions similarly to human RIG-I (hRIG-I) by mutating histidine 925 into phenylalanine, a key residue for hRIG-I RNA binding loop function, or by swapping the CTD of cMDA5 with that of hRIG-I or duck RIG-I (dRIG-I). The engineered cMDA5 gene was expressed in cMDA5 knockout DF-1 cells, and interferon-beta (IFN-ß) activity and expression of interferon-related genes were measured after transfection of cells with RNA ligands of hRIG-I or human MDA5 (hMDA5). We found that both mutant cMDA5 and engineered cMDA5 triggered significantly stronger interferon-mediated immune responses than wild-type cMDA5. Moreover, engineered cMDA5 reduced the IAV titer by 100-fold compared with that in control cells. Collectively, engineered cMDA5/RIG-I CTD significantly enhanced interferon-mediated immune responses, making them invaluable strategies for production of IAV-resistant chickens. KEY POINTS: • Mutant chicken MDA5 with critical residue of RIG-I (phenylalanine) enhanced immunity. • Engineered chicken MDA5 with CTD of RIG-I increased IFN-mediated immune responses. • Engineered chicken MDA5 reduced influenza A virus titers by up to 100-fold.

Targeted Knockout of MDA5 and TLR3 in the DF-1 Chicken Fibroblast Cell Line Impairs Innate Immune Response Against RNA Ligands.

The innate immune system, which senses invading pathogens, plays a critical role as the first line of host defense. After recognition of foreign RNA ligands (e.g., RNA viruses), host cells generate an innate immune or antiviral response via the interferon-mediated signaling pathway. Retinoic acid-inducible gene I (RIG-1) acts as a major sensor that recognizes a broad range of RNA ligands in mammals; however, chickens lack a RIG-1 homolog, meaning that RNA ligands should be recognized by other cellular sensors such as melanoma differentiation-associated protein 5 (MDA5) and toll-like receptors (TLRs). However, it is unclear which of these cellular sensors compensates for the loss of RIG-1 to act as the major sensor for RNA ligands. Here, we show that chicken MDA5 (cMDA5), rather than chicken TLRs (cTLRs), plays a pivotal role in the recognition of RNA ligands, including poly I:C and influenza virus. First, we used a knockdown approach to show that both cMDA5 and cTLR3 play roles in inducing interferon-mediated innate immune responses against RNA ligands in chicken DF-1 cells. Furthermore, targeted knockout of cMDA5 or cTLR3 in chicken DF-1 cells revealed that loss of cMDA5 impaired the innate immune responses against RNA ligands; however, the responses against RNA ligands were retained after loss of cTLR3. In addition, double knockout of cMDA5 and cTLR3 in chicken DF-1 cells abolished the innate immune responses against RNA ligands, suggesting that cMDA5 is the major sensor whereas cTLR3 is a secondary sensor. Taken together, these findings provide an understanding of the functional role of cMDA5 in the recognition of RNA ligands in chicken DF-1 cells and may facilitate the development of an innate immune-deficient cell line or chicken model.

Differential Mechanism of ATP Production Occurs in Response to Succinylacetone in Colon Cancer Cells.

Our aim was to verify the potential ability of succinylacetone (SA) to inhibit mitochondrial function, thereby suppressing cancer cell proliferation. SA treatment caused apoptosis in HCT116 and HT29 cells, but not in SW480 cells, with mitochondria playing a key role. We checked for dysfunctional mitochondria after SA treatment. Mitochondria of HT29 cells were swollen, indicating damage, whereas in HCT116 cells, several mitochondria had a diminished size. Damaged mitochondria decreased ATP production and induced reactive oxygen species (ROS) in the cells. To understand SA-induced reduction in ATP production, we investigated the electron transfer chains (ETC) and pyruvate dehydrogenase kinase (PDK) activity, which prevents the transfer of acetyl-CoA to the TCA (tricarboxylic acid) cycle by inhibiting PDH (pyruvate dehydrogenase) activity. In each cell line, the inhibitory mechanism of ATP by SA was different. The activity of complex III consisting of the mitochondrial ETCs in HT29 cells was decreased. In contrast, PDH activity in HCT116 cells was reduced. Nicotinamide nucleotide transhydrogenase (NNT)-removing reactive oxygen species (ROS) was upregulated in HT29 cells, but not in HCT116 cells, indicating that in HT29 cells, a defense mechanism was activated against ROS. Collectively, our study showed a differential mechanism occurs in response to SA in colon cancer cells.

Bisdemethoxycurcumin Induces apoptosis in activated hepatic stellate cells via cannabinoid receptor 2.

Activated Hepatic Stellate Cells (HSCs), major fibrogenic cells in the liver, undergo apoptosis when liver injuries cease, which may contribute to the resolution of fibrosis. Bisdemethoxycurcumin (BDMC) is a natural derivative of curcumin with anti-inflammatory and anti-cancer activities. The therapeutic potential of BDMC in hepatic fibrosis has not been studied thus far in the context of the apoptosis in activated HSCs. In the current study, we compared the activities of BDMC and curcumin in the HSC-T6 cell line and demonstrated that BDMC relatively induced a potent apoptosis. BDMC-induced apoptosis was mediated by a combinatory inhibition of cytoprotective proteins, such as Bcl2 and heme oxygenase-1 and increased generation of reactive oxygen species. Intriguingly, BDMC-induced apoptosis was reversed with co-treatment of sr144528, a cannabinoid receptor (CBR) 2 antagonist, which was confirmed with genetic downregulation of the receptor using siCBR2. Additionally, incubation with BDMC increased the formation of death-induced signaling complex in HSC-T6 cells. Treatment with BDMC significantly diminished total intracellular ATP levels and upregulated ATP inhibitory factor-1. Collectively, the results demonstrate that BDMC induces apoptosis in activated HSCs, but not in hepatocytes, by impairing cellular energetics and causing a downregulation of cytoprotective proteins, likely through a mechanism that involves CBR2.