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Heat is a cardinal feature of inflammation. Despite temperature variability and dependence of enzymes and complexes, how heat and fever affect immune cells remains uncertain. We found that heat broadly increased inflammatory activity of CD4+T cell subsets and decreased Treg suppressive function. Th1 cells, however, also selectively developed mitochondrial dysfunction with high levels of ROS production and DNA damage. This led Th1 cells to undergoTp53-dependent death, which was required to minimize the accumulation of mutations in heat and inflammation. Th1 cells with similar DNA damage signatures were also detected in Crohn’s disease and rheumatoid arthritis. Fever and inflammation-associated heat thus selectively induce mitochondrial stress and DNA damage in activated Th1 cells that requires p53 to maintain genomic integrity of the T cell repertoire.One Sentence SummaryFever temperatures augment CD4+T cell-mediated inflammation but induce differential metabolic stress and DNA damage in T cell subsets, with Th1 cells selectively sensitive and dependent on p53 to induce apoptosis and maintain genomic integrity.

Topological stress can cause replication forks to stall as they converge upon one another during termination of vertebrate DNA synthesis. However, replication forks ultimately overcome topological stress and complete DNA synthesis, suggesting that alternative mechanisms can overcome topological stress. We performed a proteomic analysis of converging replication forks that were stalled by topological stress in Xenopus egg extracts. We found that the helicase RTEL1 and the replisome protein MCM10 were highly enriched on DNA under these conditions. We show that RTEL1 normally plays a minor role during fork convergence while the role of MCM10 is normally negligible. However, RTEL1 and MCM10 both become crucially important for fork convergence under conditions of topological stress. RTEL1 and MCM10 exert non-additive effects on fork convergence and physically interact, suggesting that they function together. Furthermore, RTEL1 and MCM10 do not impact topoisomerase activity but do promote fork progression through a replication barrier. Thus, RTEL1 and MCM10 appear to play a general role in promoting progression of stalled forks, including when forks stall during termination. Overall, our data identify an alternate mechanism of termination involving RTEL1 and MCM10 that can be used to complete DNA synthesis under conditions of topological stress.

T cell metabolism is dynamic and highly regulated. While the intrinsic metabolic programs of T cell subsets are integral to their distinct differentiation and functional patterns, the ability of cells to acquire nutrients and cope with hostile microenvironments can limit these pathways. T cells must function in a wide variety of tissue settings, and how T cells interpret these signals to maintain an appropriate metabolic program for their demands or if metabolic mechanisms of immune suppression restrain immunity is an area of growing importance. Both in inflamed and cancer tissues, a wide range of changes in physical conditions and nutrient availability are now acknowledged to shape immunity. These include fever and increased temperatures, depletion of critical micro and macro-nutrients, and accumulation of inhibitory waste products. Here we review several of these factors and how the tissue microenvironment both shapes and constrains immunity.

SUMMARYAntigenic stimulation promotes T cells metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme Methylenetetrahydrofolate Dehydrogenase-2 (MTHFD2) is highly expressed in inflammatory diseases and induced in activated T cells to promote proliferation and produce inflammatory cytokines. In pathogenic Th17 cells, MTHFD2 also prevented aberrant upregulation of FoxP3 and suppressive capacity. Conversely, MTHFD2-deficiency enhanced lineage stability of regulatory T (Treg) cells. Mechanistically, MTHFD2 maintained cellular 10-formyltetrahydrofolate for de novo purine synthesis and MTHFD2 inhibition led to accumulation of the intermediate 5-aminoimidazole carboxamide ribonucleotide that was associated with decreased mTORC1 signaling. MTHFD2 was also required for proper histone de-methylation in Th17 cells. Importantly, inhibiting MTHFD2 in vivo reduced disease severity in Experimental Autoimmune Encephalomyelitis and Delayed-Type Hypersensitivity. MTHFD2 induction is thus a metabolic checkpoint for pathogenic effector cells that suppresses anti-inflammatory Treg cells and is a potential therapeutic target within 1C metabolism.

Peripheral myelin protein 22 (PMP22) folds and trafficks inefficiently, with only 20% of newly expressed protein trafficking to the cell surface. This behavior is exacerbated in many of the mutants associated with Charcot–Marie–Tooth disease, motivating further study. Here we characterized the role of N-glycosylation in limiting PMP22 trafficking. We first eliminated N-glycosylation using an N41Q mutation, which resulted in an almost 3-fold increase in trafficking efficiency of wildtype (WT) PMP22 and a 10-fold increase for the severely unstable L16P disease mutant in HEK293 cells, with similar results in Schwann cells. Total cellular levels were also much higher for the WT/N41Q mutant, although not for the L16P/N41Q form. Depletion of oligosaccharyltransferase OST-A and OST-B subunits revealed that WT PMP22 is N-glycosylated posttranslationally by OST-B, whereas L16P is cotranslationally glycosylated by OST-A. Quantitative proteomic screens revealed similarities and differences in the interactome for WT, glycosylation-deficient, and unstable mutant forms of PMP22 and also suggested that L16P is sequestered at earlier stages of endoplasmic reticulum quality control. CRISPR knockout studies revealed a role for retention in endoplasmic reticulum sorting receptor 1 (RER1) in limiting the trafficking of all three forms, for UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1) in limiting the trafficking of WT and L16P but not N41Q, and calnexin (CNX) in limiting the trafficking of WT and N41Q but not L16P. This work shows that N-glycosylation is a limiting factor to forward trafficking PMP22 and sheds light on the proteins involved in its quality control.

Summary Antigenic stimulation promotes T cell metabolic reprogramming to meet increased biosynthetic, bioenergetic, and signaling demands. We show that the one-carbon (1C) metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) regulates de novo purine synthesis and signaling in activated T cells to promote proliferation and inflammatory cytokine production. In pathogenic T helper-17 (Th17) cells, MTHFD2 prevented aberrant upregulation of the transcription factor FoxP3 along with inappropriate gain of suppressive capacity. MTHFD2 deficiency also promoted regulatory T (Treg) cell differentiation. Mechanistically, MTHFD2 inhibition led to depletion of purine pools, accumulation of purine biosynthetic intermediates, and decreased nutrient sensor mTORC1 signaling. MTHFD2 was also critical to regulate DNA and histone methylation in Th17 cells. Importantly, MTHFD2 deficiency reduced disease severity in multiple in vivo inflammatory disease models. MTHFD2 is thus a metabolic checkpoint to integrate purine metabolism with pathogenic effector cell signaling and is a potential therapeutic target within 1C metabolism pathways.

SUMMARY 5-Hydroxymethylcytosine (5hmC) binding, ES-cell-specific (HMCES) crosslinks to apurinic or apyrimidinic (AP, abasic) sites in single-strand DNA (ssDNA). To determine whether HMCES responds to the ssDNA abasic site in cells, we exploited the activity of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3A (APOBEC3A). APOBEC3A preferentially deaminates cytosines to uracils in ssDNA, which are then converted to abasic sites by uracil DNA glycosylase. We find that HMCES-deficient cells are hypersensitive to nuclear APOBEC3A localization. HMCES relocalizes to chromatin in response to nuclear APOBEC3A and protects abasic sites from processing into double-strand breaks (DSBs). Abasic sites induced by APOBEC3A slow both leading and lagging strand synthesis, and HMCES prevents further slowing of the replication fork by translesion synthesis (TLS) polymerases zeta (Polζ) and kappa (Polκ). Thus, our study provides direct evidence that HMCES responds to ssDNA abasic sites in cells to prevent DNA cleavage and balance the engagement of TLS polymerases., In Brief Mehta et al. use APOBEC3A to demonstrate that HMCES responds to ssDNA abasic sites in cells and prevents replication fork collapse. APOBEC3A-induced abasic sites slow both leading and lagging strand polymerization, and HMCES engagement prevents further fork slowing because of the action of TLS polymerases zeta (Polζ) and kappa (Polκ)., Graphical Abstract

SUMMARY Termination of DNA replication occurs when two replication forks converge upon the same stretch of DNA. Resolution of topological stress by topoisomerases is crucial for fork convergence in bacteria and viruses, but it is unclear whether similar mechanisms operate during vertebrate termination. Using Xenopus egg extracts, we show that topoisomerase II (Top2) resolves topological stress to prevent converging forks from stalling during termination. Under these conditions, stalling arises due to an inability to unwind the final stretch of DNA ahead of each fork. By promoting fork convergence, Top2 facilitates all downstream events of termination. Converging forks ultimately overcome stalling independently of Top2, indicating that additional mechanisms support fork convergence. Top2 acts throughout replication to prevent the accumulation of topological stress that would otherwise stall converging forks. Thus, termination poses evolutionarily conserved topological problems that can be mitigated by careful execution of the earlier stages of replication., Graphical Abstract, In Brief To complete DNA synthesis replication, forks must converge on the same stretch of DNA. In vertebrates this process occurs rapidly, but it is unclear which mechanisms support fork convergence. Heintzman et al. find that topoisomerase II promotes fork convergence by preventing accumulation of topological stress earlier during replication.