Alpha-(1,6)-fucosyltransferase (FUT8) affects the survival strategy of osteosarcoma by remodeling TNF/NF-κB2 signaling

Glycosylation is an important modification of membrane proteins that results in functional changes in many cellular activities, from cell-cell recognition to regulatory signaling. Fucosyltransferase 8 (FUT8) is the sole enzyme responsible for core fucosylation, and aberrant fucosylation by dysregulated expression of fucosyltransferases is responsible for the growth of various types of carcinomas. However, the function of FUT8 in the progress of osteosarcoma (OS) has not been reported. In this study, we found that FUT8 is expressed at lower levels in patients with OS and in human OS cell lines such as MNNG/HOS, U2OS, and 143B, suggesting that attenuated expression of FUT8 is involved in the growth and progression of OS. Mechanistically, FUT8 affects the survival strategy of OS by modifying core-fucosylation levels of TNF receptors (TNFRs). Lower fucosylation of TNFRs activates the non-canonical NF-κB signaling pathway, and in turn, decreases mitochondria-dependent apoptosis in OS cells. Together, our results point to FUT8 being a negative regulator of OS that enhances OS-cell apoptosis and suggests a novel therapeutic strategy for treating OS.

Fundamentally different roles of neuronal TNF receptors in CNS pathology: TNFR1 and IKKβ promote microglial responses and tissue injury in demyelination while TNFR2 protects against excitotoxicity in mice

Background During inflammatory demyelination, TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF (solTNF), whereas TNFR2 mediates beneficial effects of transmembrane TNF (tmTNF) through oligodendroglia, microglia, and possibly other cell types. This model supports the use of selective inhibitors of solTNF/TNFR1 as anti-inflammatory drugs for central nervous system (CNS) diseases. A potential obstacle is the neuroprotective effect of solTNF pretreatment described in cultured neurons, but the relevance in vivo is unknown. Methods To address this question, we generated mice with neuron-specific depletion of TNFR1, TNFR2, or inhibitor of NF-κB kinase subunit β (IKKβ), a main downstream mediator of TNFR signaling, and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of solTNF neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wild-type (WT) and TNF and TNFR knockout cells and measuring N-methyl-d-aspartate (NMDA) excitotoxicity in vitro. Results Neither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKβ promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone-induced demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid (KA) excitotoxicity model in mice and limited hippocampal neuron death. The protective effects of neuronal TNFR2 observed in vivo were further investigated in vitro. As previously described, pretreatment of astrocyte-neuron co-cultures with solTNF (and therefore TNFR1) protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte tmTNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist. Conclusions These results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKβ promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously described neuroprotective effects of solTNF against glutamate excitotoxicity in vitro are indirect and mediated via astrocyte tmTNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of solTNF/TNFR1 with maintenance of TNFR2 function would have combined anti-inflammatory and neuroprotective properties required for safe treatment of CNS diseases.

POS0500 NUMBER AND CO-EXPRESSION OF TNF RECEPTORS TYPE 1 AND 2 ON IMMUNOCOMPETENT CELLS ARE ASSOCIATED WITH STABILITY OF REMISSION IN RHEUMATOID ARTHRITIS PATIENTS

Background:The balance of TNFα receptors expression on cells which are actively involved in immunopathological processes affects both the density of distribution of receptors on cells and co-expression in subsets. Previously it was shown that basic effective RA therapy with methotrexate and glucocorticoids leads to equalization of the expression profile either in the percentage of cells or in the number of receptors, approaching those of healthy donors, but not simultaneously. However, questions about the relationship between the effectiveness of biological therapy and receptors co-expression remain unknown.Objectives:To assess the differences in co-expression and quantitative expression of TNF receptors type 1 and 2 in subsets of cells associated with the severity of the disease, depending on the response to rituximab therapy.Methods:Subanalysis of patients with high disease activity level successfully treated with rituximab (alone or in combination treatment scheme) during hospitalization was performed (n = 14). The first group included 6 patients who retained low disease activity during 1 month follow-up (RA, stabilization). The second group consisted of 8 patients who had exacerbation during follow-up period. As a control group, we used data from 43 comparable healthy donors. Subsets of T regulatory cells and monocytes were studied. A comparison was made among the indicators of receptors number and proportion of cells expressing the corresponding receptor.Results:For T regulatory cells, the key differences for patients who did not retain low disease activity were significantly higher number of TNF type 1 and type 2 receptors on double-positive cells with a lower percentage of these cells compared to stable patients. At the same time, higher differences between proportions of double-positive cells in comparison with control values of healthy donors were associated with higher probability of maintaining in remission.For monocytes, the key differences in stable patients were the very high quantitative expression of type 1 receptors on double-positive cells, with a lower percentage of these cells compared to patients with exacerbation. At the same time, lower differences between proportions of double-positive cells in comparison with control values of healthy donors were associated with higher probability of maintaining in remission.Conclusion:Obtained data confirm the previously proposed hypothesis about the essential role of balance in quantitative expression of TNF receptors type 1 and 2 on double-positive cells to determine the intensity and type of cell response to the mediator and its association with the level of disease activity and response to therapy.Acknowledgements:This study is supported by grant of the President of the Russian Federation for state support of young Russian PhD scientists №МК-2433.2020.4Disclosure of Interests:None declared

Selected Uterine Immune Events Associated With the Establishment of Pregnancy in the Dog

In the dog, implantation takes place at approximately 17 days of embryonal life and, while exposed to relatively high circulating progesterone concentrations, embryos presence is required for the formation of decidua. Furthermore, a balance between pro- and anti-inflammatory responses in conceptus-maternal communication is crucial for the onset of pregnancy. Strikingly, the understanding of such immune mechanisms in canine reproduction is still elusive. Here, canine uterine samples from pre-implantation (day 10–12, E+) and corresponding non-pregnant controls (E–), implantation (day 17, Imp) and post-implantation (day 18–25, Post-Imp) stages of pregnancy were used to investigate the expression and localization of several immune-related factors. The most important findings indicate increased availability of CD4, MHCII, NCR1, IDO1, AIF1, CD25, CCR7, and IL6 in response to embryo presence (E+), while FoxP3 and CCL3 were more abundant in E– samples. Implantation was characterized by upregulated levels of FoxP3, IL12a, ENG, and CDH1, whereas CD4, CCR7, IL8, and -10 were less represented. Following implantation, decreased transcript levels of TNFR1, MHCII, NCR1, TLR4, CD206, FoxP3, and IL12a were observed concomitantly with the highest expression of IL6 and IL1β. MHCII, CD86, CD206, CD163, TNFα, IDO1, and AIF1 were immunolocalized in macrophages, CD4 and Nkp46 in lymphocytes, and some signals of IDO1, AIF1, and TNF-receptors could also be identified in endothelial cells and/or uterine glands. Cumulatively, new insights regarding uterine immunity in the peri-implantation period are provided, with apparent moderated pro-inflammatory signals prevailing during pre-implantation, while implantation and early trophoblast invasion appear to be associated with immunomodulatory and rather anti-inflammatory conditions.

Fundamentally Different Roles of Neuronal TNF Receptors in CNS Pathology: TNFR1 and IKKβ Promote Microglial Responses and Tissue Injury in Demyelination, TNFR2 Protects in Excitotoxicity in Mice

BACKGROUNDDuring inflammatory demyelination TNF receptor 1 (TNFR1) mediates detrimental proinflammatory effects of soluble TNF, whereas TNFR2 mediates beneficial effects of transmembrane TNF through oligodendrocytes, microglia, and possibly other cell types. This model supports use of selective inhibitors of soluble TNF/TNFR1 as antinflammatory drugs for CNS disease. A potential obstacle is the neuroprotective effect of soluble TNF pretreatment described in cultured neurons, but the in vivo relevance is unknown. METHODSTo address this question we generated mice with neuron-specific depletion of TNFR1, TNFR2 or IKKβ and applied experimental models of inflammatory demyelination and acute and preconditioning glutamate excitotoxicity. We also investigated the molecular and cellular requirements of soluble TNF (and therefore TNFR1) neuroprotection by generating astrocyte-neuron co-cultures with different combinations of wildtype and TNF and TNF receptor knockout cells and measuring NMDA excitotoxicity in vitro.RESULTSNeither neuronal TNFR1 nor TNFR2 protected mice during inflammatory demyelination. In fact, both neuronal TNFR1 and neuronal IKKβ promoted microglial responses and tissue injury, and TNFR1 was further required for oligodendrocyte loss and axonal damage in cuprizone demyelination. In contrast, neuronal TNFR2 increased preconditioning protection in a kainic acid excitotoxicity model in mice, and limited hippocampal neuron death. The neuroprotective effects of neuronal TNFR2 observed in vivo were further investigated in vitro. Here as expected, pretreatment of astrocyte-neuron co-cultures with soluble TNF protected them against NMDA excitotoxicity. However, protection was dependent on astrocyte, not neuronal TNFR1, on astrocyte transmembrane TNF-neuronal TNFR2 interactions, and was reproduced by a TNFR2 agonist. CONCLUSIONSThese results demonstrate that neuronal TNF receptors perform fundamentally different roles in CNS pathology in vivo, with neuronal TNFR1 and IKKβ promoting microglial inflammation and neurotoxicity in demyelination, and neuronal TNFR2 mediating neuroprotection in excitotoxicity. They also reveal that previously-described neuroprotective effects of soluble TNF (and therefore TNFR1) against glutamate excitotoxicity in vitro are indirect, and mediated by astrocyte transmembrane TNF-neuron TNFR2 interactions. These results consolidate the concept that selective inhibition of soluble TNF/TNFR1 with maintenance of TNFR2 function would have anti-inflammatory and neuroprotective properties required for the safe treatment of CNS disease.