Numbers of patients with coronavirus disease 2019 (COVID-19) have increased rapidly worldwide. Plasma levels of full-length galectin-9 (FL-Gal9) and osteopontin (FL-OPN) as well as their truncated forms (Tr-Gal9, Ud-OPN, respectively), are representative inflammatory biomarkers. Here, we measured FL-Gal9, FL-OPN, Tr-Gal9, and Ud-OPN in 94 plasma samples obtained from 23 COVID-19-infected patients with mild clinical symptoms (CV), 25 COVID-19 patients associated with pneumonia (CP), and 14 patients with bacterial infection (ID). The four proteins were significantly elevated in the CP group when compared with healthy individuals. ROC analysis between the CV and CP groups showed that C-reactive protein had the highest ability to differentiate, followed by Tr-Gal9 and ferritin. Spearman’s correlation analysis showed that Tr-Gal9 and Ud-OPN but not FL-Gal9 and FL-OPN, had a significant association with laboratory markers for lung function, inflammation, coagulopathy, and kidney function in CP patients. CP patients treated with tocilizumab had reduced levels of FL-Gal9, Tr-Gal9, and Ud-OPN. It was suggested that OPN is cleaved by interleukin-6-dependent proteases. These findings suggest that the cleaved forms of OPN and galectin-9 can be used to monitor the severity of pathological inflammation and the therapeutic effects of tocilizumab in CP patients.
Extracellular Galectins constitute a novel mechanism of membrane protein organisation at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organisation and function. By combining functional, super-resolution and atomic force microscopy experiments to analyse membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure via modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organisation. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type lectin receptor-mediated pathogen uptake by human DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionary conserved.
Osteopontin (OPN) mediates bone remodeling and tissue debridement. The OPN protein is cleaved, but it is unclear how full-length (FL)-OPN or its cleaved form perform their biological activities in target cells. We, therefore, performed the molecular characterization of OPN in exosomes (Exo). The Exo were isolated from lipopolysaccharide (LPS)-stimulated phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages. The Exo were also isolated from PMA-differentiated THP-1 macrophages. The Exo were identified using the qNano multiple analyzer (diameter 59–315 nm) and western blotting with a CD9 antibody. LPS-stimulated cells produced more particles than non-stimulated cells. The presence of the FL or the cleaved form of OPN was confirmed using western blot analysis. A mixture of FL and cleaved OPN was also measured using an ELISA system (Ud-OPN) and their presence in the Exo was confirmed. Ud/FL ratios became low after LPS stimulation, indicating the enhanced encapsulation of FL-OPN in the Exo by LPS. These findings suggest that LPS stimulation of human macrophages facilitates the synthesis of FL-OPN, which is cleaved in cells or the Exo after release. These findings indicate that Exo is a suitable vehicle to transfer OPN to the target cells.
Galectin-9 (Gal-9) is known to contribute to antiviral responses in coronavirus disease 2019 (COVID-19). Increased circulating Gal-9 in COVID-19 is associated with COVID-19 severity. In a while, the linker-peptide of Gal-9 is susceptible to proteolysis that can cause the change or loss of Gal-9 activity. Here, we measured plasma levels of N-cleaved-Gal9, which is Gal9 carbohydrate-recognition domain at the N-terminus (NCRD) with attached truncated linker peptide that differs in length depending on the type of proteases, in COVID-19. We also investigated the time course of plasma N-cleaved-Gal9 levels in severe COVID-19 treated with tocilizumab (TCZ).. As a result, we observed an increase in plasma N-cleaved-Gal9 levels in COVID-19 and its higher levels in COVID-19 with pneumonia compared to the mild cases (healthy: 326.1 pg/mL, mild: 698.0 pg/mL, and with pneumonia: 1570 pg/mL). N-cleaved-Gal9 levels were associated with lymphocyte counts,C-reactive protein (CRP), soluble interleukin-2 receptor (sIL-2R), D-dimer, and ferritin levels, and ratio of percutaneous oxygen saturation to fraction of inspiratory oxygen (S/F ratio) in COVID-19 with pneumonia and discriminated different severity groups with high accuracy (area under the curve (AUC): 0.9076). Both N-cleaved-Gal9 and sIL-2R levels were associated with plasma matrix metalloprotease (MMP)-9 levels in COVID-19 with pneumonia. Furthermore, a decrease in N-cleaved-Gal9 levels was associated with a decrease of sIL-2R levels during TCZ treatment. N-cleaved-Gal9 levels showed a moderate accuracy (AUC: 0.8438) for discriminating the period before TCZ from the recovery phase. These data illustrate that plasma N-cleaved-Gal9 is a potential surrogate marker for assessing COVID-19 severity and the therapeutic effects of TCZ.
Following a dramatic rise in incidence in recent decades, Dengue virus infection (DENV) is now a significant burden of disease in the tropics and major international public health concern. Currently no treatment or effective vaccine exists. The β-Galactoside-binding lectin, Galectin-9 (Gal-9), functions as pro-apoptotic agent and inducer of pro-inflammatory cytokines from T cells. Here, we sought to evaluate the role of Gal-9 in the pathogenesis of acute DENV in patients presenting with self-limiting dengue fever (DF) and more serious life-threatening dengue hemorrhagic fever (DHF). Blood samples were obtained from 65 patients (mean age: 23±4yrs) hospitalized with DENV from the San Lazaro Hospital, Manila, Philippines in 2010. Plasma Gal-9 was measured by an ELISA method at the critical phase (day4-5) and recovery phase (day7-8) of infection. Our results show that during the critical phase, Gal-9 levels were markedly elevated in DF (1758±1047pg/ml) and DHF(2730±1848pg/ml) compared to uninfected (363±280pg/ml) (p<0.05) controls. These levels of Gal-9 were the highest ever observed in humans. During the recovery phase, Gal-9 levels significantly declined (1170pg/ml and 1140pg/ml) (p<0.05) from peak levels in DF and DHF, respectively, but not to levels observed in uninfected donors. The above findings suggest that Gal-9 levels tracks disease severity during acute DEN, potentially mediate immuno-pathology associated with DHF, and may serve as an indicator of recovery from DENV.
Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin which is frequently released in the extra-cellular medium where it can modulate various biological activities (cell adhesion, migration), but it acts mainly as a negative regulator of the immune response. Our team has shown an intense and inappropriate production of gal-9 by malignant epithelial cells in Epstein-Barr virus-associated nasopharyngeal carcinoma. Other groups and ourselves have shown that gal-9 is very abundant in plasma samples from patients chronically infected by hepatitis C or B virus, and particularly in those with hepatocellular carcinomas. The most prominent immunosuppressive effects reported for gal-9 are the induction of apoptosis of CD4+ T-helper 1 (Th1) cells, exhaustion of CD8+ T cells, and stimulation of regulatory T cell activity. However, a recent study (and our own data) demonstrates that gal-9 can also activate and expand a subset of IFNγ; producing Th1 cells and central memory T cells in the surviving population. Despite its critical role in regulating the immune response, our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. Our studies reveal that several proximal components of the TCR-CD3 complex including the Lck kinase were required for the calcium (Ca2+) mobilization induced by gal-9 in Jurkat cells. In contrast, these components were not necessary for the apoptosis pathway triggered by gal-9. These data were confirmed in human CD4+ blood T cells: Lck inhibition with a specific inhibitor abrogates Ca2+ mobilization, but not apoptosis induction in CD4+ T cells. Moreover, this cytosolic Ca2+ release leads to the production of Th1 type cytokines, i.e. IL-2 and IFNγ; also dependent on Lck. These findings support the notion that gal-9 acts on T cells by two distinct pathways: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis which is independent of this complex. More recent data suggest that the CD45 molecule could be a putative receptor for gal-9 on human T cells. Indeed, the J45.01 cell line (CD45-deficient) was not susceptible to gal-9-induced apoptosis. In parallel to this work, we investigated the same cellular events (apoptosis and Ca2+ mobilization) in human B cells. In fact, there are currently very few publications dealing with the effects of gal-9 on B cells. Our preliminary results show that some Burkitt's lymphoma cell lines (BL2, BL41 and Ramos) and CD20+ B cells from blood, were both sensitive to gal-9-induced apoptosis. Besides, gal-9 induced a cytosolic Ca2+ mobilization in BL2 and BL41 cells which is efficiently abrogated by the chemical Lck inhibitor. In contrast, we didn't observe any Ca2+ release in the Ramos cell line, in which the Lck kinase is not functional, as well as in non-lymphoid cells. These preliminary results suggest that gal-9 activates in B cells a signaling pathway somehow similar to the TCR pathway and previously unexplored in B cells. Citation Format: Claire Lhuillier, Clement Barjon, Toshiro Niki, Aurore Gelin, Nadira Delhem, Mitsuomi Hirashima, Ming Wei, Olivier Dellis, Pierre Busson. Exploration of the signaling pathways triggered by exogenous galectin-9 in human T and B cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B177.
4-1BB is an inducible stimulatory molecule in the TNFR superfamily expressed on activated T cells, dendritic cells, NK cells, and other lymphoid cell types. Although known to bind the TNF family molecule 4-1BBL, we searched for novel binding partners that might modulate or control the activity of 4-1BB. We found that 4-1BB can bind to Galectin-9, a member of the beta-galactoside binding lectins containing homologous carbohydrate recognition domains (CRDs). Surface plasmon resonance analysis for mouse and human molecules demonstrated that the equilibrium binding constant (KD) of this interaction was very strong at ~11 nM and ~85 nM respectively. Functional studies in vitro showed that the stimulatory activity of 4-1BB was dependent on Galectin-9 in that Galectin-9-/- NK cells, CD4 T cells, or CD8 T cells, were largely unresponsive when 4-1BB was ligated but had normal activity when other stimulatory receptors were engaged. Similarly, 4-1BB activity on dendritic cells was impaired when Galectin-9 was absent. Additionally, in EAE and Asthma models, where an agonist of 4-1BB can suppress disease via induction of CD8β+CD11c+ regulatory cells, we found that this protective effect of anti-4-1BB was lost in Galectin-9-/- mice. Thus, our data demonstrate a novel function of Galectin-9 in facilitating signaling through 4-1BB, and suggest that Galectin-9 may allow 4-1BB to oligomerize and/or cluster in a manner that allows efficient engagement of intracellular signaling pathways.
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