Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN- production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46- and C3-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN (the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)) as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity – but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn–/– mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an 'upstream' activator of the CTSL-C3-IFN-γ axis in human CD4+ T cells and hence an important supporter of human Th1 induction.
While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the “complosome,” functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also nonredundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux toward reactive oxygen species generation and anaerobic glycolysis to favor IL-1β production, both at the transcriptional level and processing of pro–IL-1β. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1β produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell-autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.
Expansion and acquisition of Th1 cell effector function requires metabolic reprogramming; however, the signals instructing these adaptations remain poorly defined. Here we found that in activated human T cells, autocrine stimulation of the complement receptor CD46, and specifically its intracellular domain CYT-1, was required for induction of the amino acid (AA) transporter LAT1 and enhanced expression of the glucose transporter GLUT1. Furthermore, CD46 activation simultaneously drove expression of LAMTOR5, which mediated assembly of the AA-sensing Ragulator-Rag-mTORC1 complex and increased glycolysis and oxidative phosphorylation (OXPHOS), required for cytokine production. T cells from CD46-deficient patients, characterized by defective Th1 cell induction, failed to upregulate the molecular components of this metabolic program as well as glycolysis and OXPHOS, but IFN-γ production could be reinstated by retrovirus-mediated CD46-CYT-1 expression. These data establish a critical link between the complement system and immunometabolic adaptations driving human CD4+ T cell effector function.
Abstract Intracellular/autocrine complement proteins have emerged as critical regulators of human Th1 induction and contraction. T cells contain both intracellular C3 and C5 activation systems, with intracellular C3aR1 and C5aR1 stimulation driving T cell homeostatic survival and normal Th1 IFNg production, respectively. Here we demonstrate how the intracellular/autocrine C5 system is regulated by using T cells from the first described patient with C5aR2 deficiency. This patient suffers from an autoinflammatory syndrome, with enhanced inflammatory Th responses and a profound loss of naïve CD4 T cells in the blood (95% have a memory phenotype). Thus, in contrast to intracellular C5aR1 stimulation, cell surface expressed C5aR2 is an important negative regulator of inflammatory Th induction. While both C5aR1 and C5aR2 can bind C5a, we found that the carboxypeptidase-processed form of C5a, C5a-desArg, was twice as potent as C5a in reducing Th1 induction. In addition, carboxypeptidase M (CPM) expression was highly induced upon T cell activation indicating that CPM may be mediating T cell-derived C5a-desArg generation and C5aR2 stimulation. In this vein, activation of CRISPR/Cas9 generated CPMKO T cells, or of T cells in the presence of a CPM inhibitor, induced Th1 hyper-induction, which was rescued by addition of a C5aR2 agonist and reduced by adding C5a-desArg, but not C5a. The in vivo importance of T cell-expressed CPM and autocrine C5aR2 activation is demonstrated by the enhanced inflammatory Th responses in Cpm −/−mice and increased pathology caused by Cpm −/−T cells in a T cell transfer colitis model. These data highlight the importance of intracellular/autocrine C5 system regulation by CPM through C5aR2 signaling in T cell responses.
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