Increased IL-2 and reduced TGF-β upon T-cell stimulation are associated with GM-CSF upregulation in multiple immune cell types in multiple sclerosis

© 2020 by the authors. Granulocyte macrophage colony stimulating factor (GM-CSF) is a pro-inflammatory cytokine produced by immune cells. Recent evidence suggests that GM-CSF plays an important role in multiple sclerosis (MS) pathogenesis. We investigated the expression and regulation of GM-CSF in different immune cells in MS. We also investigated the differentiation and frequency of GM-CSF-producing Th cells that do not co-express interferon (IFN)-γ or interleukin-17 (IL-17) (Th-GM cells) in MS. We found a significant increase in the percentage of GM-CSF-expressing Th cells, Th1 cells, Th-GM cells, cytotoxic T (Tc) cells, monocytes, natural killer (NK) cells, and B cells in PBMC from MS patients stimulated with T cell stimuli. Stimulated PBMC culture supernatants from MS patients contained significantly higher levels of IL-2, IL-12, IL-1β, and GM-CSF and significantly lower levels of transforming growth factor (TGF-)β. Blocking IL-2 reduced the frequency of Th-GM cells in PBMC from MS patients. The frequency of Th-GM cells differentiated in vitro from naive CD4+ T cells was significantly higher in MS patients and was further increased in MS with IL-2 stimulation. These findings suggest that all main immune cell subsets produce more GM-CSF in MS after in vitro stimulation, which is associated with defective TGF-β and increased IL-2 and IL-12 production. Th-GM cells are increased in MS. GM-CSF may be a potential therapeutic target in MS

Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes

Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 which controls G1-to-S-phase transition by targeting degradation motifs, notably the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture. We now show that APC/C-Cdh1 controls the proliferative response of human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating T lymphocytes. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression

Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells

During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/cyclosome–Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein–β-transducin repeat-containing protein (SCF-β-TrCP), which control the transient appearance and metabolic activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S273) within the conserved recognition site for SCF-β-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-Glu-Asn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-β-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-to-late G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division