Activation of the Inflammatory Cytokine Genes by Intracellular Galectin-9 in Human Monocytes.
Authors: Matsuura Ai , Tsukada Junichi , Hirashima Mitsuomi , Tanaka Yoshiya 
Published: April 2020

Abstract
Galectins are a family member of animal lectins that function both extracellularly (by interacting with cell surface and extracellular matrix glycoproteins and glycolipids) and intracellularly (by interacting with cytoplasmic and nuclear proteins) to modulate signaling pathways. To date, 15 galectins have been cloned in mammals. The members can be classified into 3 subtypes according to their structure. The proto type and chimera type galectins have a single CRD. The tandem-repeat type galectins have two CRDs. In this regard, galectin-9 is a tandem repeat type galectin, which has been identified as a tumor antigen of unknown functions in patients with Hodgkin lymphoma. Galectin-9 has recently demonstrated to induce chemotaxis of eosinophils and apoptosis in a variety of cells, resulting in immunomodulation, inflammation and anti-metastatic potential of tumor cells. However, the role of galectin-9 in the inflammatory responses still remains unclear. In the present study, we investigated whether intracellular galectin-9 contributes to activation of the inflammatory cytokine genes such as IL-1α, IL-1β and IFNγin human monocytes. Protein expression of galectin-9 in human THP-1 monocytic cells was examined in western blot with anti-galectin-9 Ab and immunostaining studies using a confocal microscopy. A galectin-9 expression vector pBKCMV3-Galectin-9 was transiently cotransfected into THP-1 cells along with luciferase reporter plasmids for IL-1α, IL-1β and IFNγgene promoters. Galectin-9 was detected in the cytoplasm of untreated THP-1 cells, and the expression levels of galectin-9 protein was significantly enhanced in THP-1 cells treated with lipopolysaccharide (LPS). In transient transfection studies, overexpression of galectin-9 dose-dependently activated IL-1α, IL-1β and IFNγgene promoters in THP-1 cells, showing that intracellular galectin-9 has capacity to induce the inflammatory cytokine genes in monocytes. This argument was further supported by our reverse transcription-polymerase chain reaction (RT-PCR) data that galectin-9 overexpression enhanced mRNA levels of IL-1α and β in THP-1 cells. When luciferase reporter plasmids for transcription factors including NF-IL6, NF-κB and AP-1 were transiently cotransfected into THP-1 cells along with pBKCMV3-Galectin-9, NF-IL6 and AP-1 activities were induced by galectin-9 overexpression. Our coimmunoprecipitation study, using anti-galectin-9 Ab and anti-NF-IL6 Ab revealed that intracellular galectin-9 was physically associated with NF-IL6. On the other hand, galectin-9 failed to induce NF-κB activity. Additionally, in contrast to the data obtained from transient transfection studies using pBKCMV3-Galectin-9, no significant induction of IL-1α promoter activity was observed by adding galectin-9 protein to THP-1 cell culture medium, indicating that extracellular galectin-9 dose not affect the inflammatory cytokine gene regulation. Our results strongly suggested that intracellular galectin-9 has the capability to transactivate the inflammatory cytokine genes through the physical interaction with leucin zipper transcription factors such as NF-IL6 in monocytes.