Pharmacological HDAC inhibition impairs pancreatic ß-cell function through an epigenome-wide reprogramming.

Histone deacetylases enzymes (HDACs) are chromatin modifiers that regulate gene expression through deacetylation of lysine residues within specific histone and non-histone proteins. A cell-specific gene expression pattern defines the identity of insulin-producing pancreatic ß cells, yet molecular networks driving this transcriptional specificity are not fully understood. Here, we investigated the HDAC-dependent molecular mechanisms controlling pancreatic ß-cell identity and function using the pan-HDAC inhibitor trichostatin A through chromatin immunoprecipitation assays and RNA sequencing experiments. We observed that TSA alters insulin secretion associated with ß-cell specific transcriptome programming in both mouse and human ß-cell lines, as well as on human pancreatic islets. We also demonstrated that this alternative ß-cell transcriptional program in response to HDAC inhibition is related to an epigenome-wide remodeling at both promoters and enhancers. Our data indicate that HDAC activity could be required to protect against loss of ß-cell identity with unsuitable expression of genes associated with alternative cell fates.

Flow Cytometry-based Method for Efficient Sorting of Senescent Cells.

Cellular senescence is a reprogrammed cell state triggered as an adaptative response to a variety of stresses, most often those affecting the genome integrity. Senescent cells accumulate in most tissues with age and contribute to the development of several pathologies. Studying molecular pathways involved in senescence induction and maintenance, or in senescence escape, can be hindered by the heterogeneity of senescent cell populations. Here, we describe a flow cytometry strategy for sorting senescent cells according to three senescence canonical markers whose thresholds can be independently adapted to be more or less stringent: (i) the senescence-associated-ß-galactosidase (SA-ß-Gal) activity, detected using 5-dodecanoylaminofluorescein Di-ß-D-galactopyranoside (C12FDG), a fluorigenic substrate of ß-galactosidase; (ii) cell size, proportional to the forward scatter value, since increased size is one of the major changes observed in senescent cells; and (iii) cell granularity, proportional to the side scatter value, which reflects the accumulation of aggregates, lysosomes, and altered mitochondria in senescent cells. We applied this protocol to the sorting of normal human fibroblasts at the replicative senescence plateau. We highlighted the challenge of sorting these senescent cells because of their large sizes, and established that it requires using sorters equipped with a nozzle of an unusually large diameter: at least 200 µm. We present evidence of the sorting efficiency and sorted cell viability, as well as of the senescent nature of the sorted cells, confirmed by the detection of other senescence markers, including the expression of the CKI p21 and the presence of 53BP1 DNA damage foci. Our protocol makes it possible, for the first time, to sort senescent cells from contaminating proliferating cells and, at the same time, to sort subpopulations of senescent cells featuring senescent markers to different extents. Graphical abstract.

A time- and space-resolved nuclear receptor atlas in mouse liver.

The functional versatility of the liver is paramount for organismal homeostasis. Adult liver functions are controlled by a tightly regulated transcription factor network including nuclear receptors (NRs), which orchestrate many aspects of hepatic physiology. NRs are transcription factors sensitive to extracellular cues such as hormones, lipids, xenobiotics, etc. and are modulated by intracellular signaling pathways. While liver functional zonation and adaptability to fluctuating conditions rely on a sophisticated cellular architecture, a comprehensive knowledge of NR functions within liver cell populations is still lacking. As a step toward the accurate mapping of NR functions in the liver, we characterized their levels of expression in the whole liver from C57Bl6/J male mice as a function of time and diet. Nr1d1 (Rev-erba), Nr1d2 (Rev-erbb), Nr1c2 (Pparb/d), and Nr1f3 (Rorg) exhibited a robust cyclical expression in ad libitum-fed mice which was, like most cyclically expressed NRs, reinforced upon time-restricted feeding. In a few instances, cyclical expression was lost or gained as a function of the feeding regimen. NR isoform expression was explored in purified hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells, and liver sinusoidal cells. The expression of some NR isoforms, such as Nr1h4 (Fxra) and Nr1b1 (Rara) isoforms, was markedly restricted to a few cell types. Leveraging liver single-cell RNAseq studies yielded a zonation pattern of NRs in hepatocytes, liver sinusoidal cells, and stellate cells, establishing a link between NR subtissular localization and liver functional specialization. In summary, we provide here an up-to-date compendium of NR expression in mouse liver in space and time.

Hematopoietic Somatic Mosaicism Is Associated With an Increased Risk of Postoperative Atrial Fibrillation.

BACKGROUND: On-pump cardiac surgery triggers sterile inflammation and postoperative complications such as postoperative atrial fibrillation (POAF). Hematopoietic somatic mosaicism (HSM) is a recently identified risk factor for cardiovascular diseases and results in a shift toward a chronic proinflammatory monocyte transcriptome and phenotype. OBJECTIVES: The aim of this study was to assess the prevalence, characteristics, and impact of HSM on preoperative blood and myocardial myeloid cells as well as on outcomes after cardiac surgery. METHODS: Blood DNA from 104 patients referred for surgical aortic valve replacement (AVR) was genotyped using the HemePACT panel (576 genes). Four screening methods were applied to assess HSM, and postoperative outcomes were explored. In-depth blood and myocardial leukocyte phenotyping was performed in selected patients using mass cytometry and preoperative and postoperative RNA sequencing analysis of classical monocytes. RESULTS: The prevalence of HSM in the patient cohort ranged from 29%, when considering the conventional HSM panel (97 genes) with variant allelic frequencies ≥2%, to 60% when considering the full HemePACT panel and variant allelic frequencies ≥1%. Three of 4 explored HSM definitions were significantly associated with higher risk for POAF. On the basis of the most inclusive definition, HSM carriers exhibited a 3.5-fold higher risk for POAF (age-adjusted OR: 3.5; 95% CI: 1.52-8.03; P = 0.003) and an exaggerated inflammatory response following AVR. HSM carriers presented higher levels of activated CD64+CD14+CD16- circulating monocytes and inflammatory monocyte-derived macrophages in presurgery myocardium. CONCLUSIONS: HSM is frequent in candidates for AVR, is associated with an enrichment of proinflammatory cardiac monocyte-derived macrophages, and predisposes to a higher incidence of POAF. HSM assessment may be useful in the personalized management of patients in the perioperative period. (Post-Operative Myocardial Incident & Atrial Fibrillation [POMI-AF]; NCT03376165).

The out-of-field dose in radiation therapy induces delayed tumorigenesis by senescence evasion.

A rare but severe complication of curative-intent radiation therapy is the induction of second primary cancers. These cancers preferentially develop not inside the planning target volume (PTV) but around, over several centimeters, after a latency period of 1-40 years. We show here that normal human or mouse dermal fibroblasts submitted to the out-of-field dose scattering at the margin of a PTV receiving a mimicked patient's treatment do not die but enter in a long-lived senescent state resulting from the accumulation of unrepaired DNA single-strand breaks, in the almost absence of double-strand breaks. Importantly, a few of these senescent cells systematically and spontaneously escape from the cell cycle arrest after a while to generate daughter cells harboring mutations and invasive capacities. These findings highlight single-strand break-induced senescence as the mechanism of second primary cancer initiation, with clinically relevant spatiotemporal specificities. Senescence being pharmacologically targetable, they open the avenue for second primary cancer prevention.

IFNγ-producing NK cells in adipose tissue are associated with hyperglycemia and insulin resistance in obese women.

BACKGROUND/OBJECTIVES: Innate lymphoid cells (ILCs) play an important role in the maintenance of immune and metabolic homeostasis in adipose tissue (AT). The crosstalk between AT ILCs and adipocytes and other immune cells coordinates adipocyte differentiation, beiging, glucose metabolism and inflammation. Although the metabolic and homeostatic functions of mouse ILCs have been extensively investigated, little is known about human adipose ILCs and their roles in obesity and insulin resistance (IR). SUBJECTS/METHODS: Here we characterized T and NK cell populations in omental AT (OAT) from women (n = 18) with morbid obesity and varying levels of IR and performed an integrated analysis of metabolic parameters and adipose tissue transcriptomics. RESULTS: In OAT, we found a distinct population of CD56-NKp46+EOMES+ NK cells characterized by expression of cytotoxic molecules, pro-inflammatory cytokines, and markers of cell activation. AT IFNγ+ NK cells, but not CD4, CD8 or γδ T cells, were positively associated with glucose levels, glycated hemoglobin (HbA1c) and IR. AT NK cells were linked to a pro-inflammatory gene expression profile in AT and developed an effector phenotype in response to IL-12 and IL-15. Moreover, integrated transcriptomic analysis revealed a potential implication of AT IFNγ+ NK cells in controlling adipose tissue inflammation, remodeling, and lipid metabolism. CONCLUSIONS: Our results suggest that a distinct IFNγ-producing NK cell subset is involved in metabolic homeostasis in visceral AT in humans with obesity and may be a potential target for therapy of IR.

Deletion of the nuclear receptor RORα in macrophages does not modify the development of obesity, insulin resistance and NASH.

Retinoic acid receptor-related orphan receptor-alpha (RORα) is a transcription factor from the nuclear receptor family expressed by immune cells and involved in the development of obesity, insulin resistance (IR) and non-alcoholic steatohepatitis (NASH). It was recently reported that mice deficient for RORα in macrophages develop more severe NASH upon high fat diet (HFD) feeding due to altered Kupffer cell function. To better understand the role of RORα in obesity and IR, we independently generated a macrophage RORα-deficient mouse line. We report that RORα deletion in macrophages does not impact on HFD-induced obesity and IR. Surprisingly, we did not confirm an effect on NASH development upon HFD feeding nor in the more severe and obesity-independent choline-deficient, L-amino acid-defined diet model. Our results therefore show that RORα deletion in macrophages does not alter the development of obesity and IR and question its role in NASH.

Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver.

Liver injury triggers adaptive remodeling of the hepatic transcriptome for repair/regeneration. We demonstrate that this involves particularly profound transcriptomic alterations where acute induction of genes involved in handling of endoplasmic reticulum stress (ERS) is accompanied by partial hepatic dedifferentiation. Importantly, widespread hepatic gene downregulation could not simply be ascribed to cofactor squelching secondary to ERS gene induction, but rather involves a combination of active repressive mechanisms. ERS acts through inhibition of the liver-identity (LIVER-ID) transcription factor (TF) network, initiated by rapid LIVER-ID TF protein loss. In addition, induction of the transcriptional repressor NFIL3 further contributes to LIVER-ID gene repression. Alteration to the liver TF repertoire translates into compromised activity of regulatory regions characterized by the densest co-recruitment of LIVER-ID TFs and decommissioning of BRD4 super-enhancers driving hepatic identity. While transient repression of the hepatic molecular identity is an intrinsic part of liver repair, sustained disequilibrium between the ERS and LIVER-ID transcriptional programs is linked to liver dysfunction as shown using mouse models of acute liver injury and livers from deceased human septic patients.

Metabolic and Innate Immune Cues Merge into a Specific Inflammatory Response via the UPR.

Innate immune responses are intricately linked with intracellular metabolism of myeloid cells. Toll-like receptor (TLR) stimulation shifts intracellular metabolism toward glycolysis, while anti-inflammatory signals depend on enhanced mitochondrial respiration. How exogenous metabolic signals affect the immune response is unknown. We demonstrate that TLR-dependent responses of dendritic cells (DCs) are exacerbated by a high-fatty-acid (FA) metabolic environment. FAs suppress the TLR-induced hexokinase activity and perturb tricarboxylic acid cycle metabolism. These metabolic changes enhance mitochondrial reactive oxygen species (mtROS) production and, in turn, the unfolded protein response (UPR), leading to a distinct transcriptomic signature with IL-23 as hallmark. Interestingly, chemical or genetic suppression of glycolysis was sufficient to induce this specific immune response. Conversely, reducing mtROS production or DC-specific deficiency in XBP1 attenuated IL-23 expression and skin inflammation in an IL-23-dependent model of psoriasis. Thus, fine-tuning of innate immunity depends on optimization of metabolic demands and minimization of mtROS-induced UPR.

Treg depletion followed by intracerebral CpG-ODN injection induce brain tumor rejection.

Using brain lymphoma model, we demonstrate that immunotherapy combining Treg depletion (using anti-CD25 mAb PC61) followed by intracranial CpG-ODN administration induced tumor rejection in all treated mice and led to the establishment of a memory antitumor immune response in 60% of them. This protective effect was associated with a recruitment of NK cells and, to a lesser extent, of dendritic cells, B cells and T lymphocytes. NK cell depletion abolished the protective effect of the treatment, confirming a major role of NK cells in brain tumor elimination. Each treatment used alone failed to protect brain tumor bearing mice, revealing the therapeutic benefit of combining Treg depletion and local CpG-ODN injection.

Efficiently stimulated adult microglia cross-prime naive CD8+ T cells injected in the brain.

Microglia are the major myeloid-immune cells of the brain parenchyma. In a steady state, microglia monitor their environment for pathogens or damaged cells. In response to neural injury or inflammation, microglia become competent APCs able to prime CD4(+) and CD8(+) T lymphocytes. We previously demonstrated that neonatal and adult microglia cross-present exogenous soluble Ags in vitro. However, whether microglia are able to cross-present Ag to naive CD8(+) T cells in vivo, within the brain microenvironment, remains undetermined. Here, we have designed an original protocol in order to exclude the involvement in cross-presentation activity of peripheral migrating APCs and of CNS-associated APCs. In C57Bl/6 mice, in which the body but not the head has been properly irradiated, we analyzed the ability of resident microglia to stimulate intracerebrally injected CD8(+) T cells in vivo. This study demonstrates for the first time that adult microglia cross-present Ag to naive CD8(+) T cells in vivo and that full microglia activation is required to overcome the inhibitory constrains of the brain and to render microglia able to cross-prime naive CD8(+) T cells injected in the brain. These observations offer new insights in brain-tumor immunotherapy based on the induction of cytotoxic antitumoral T cells.

Molecular mechanisms of resistance to 5-fluorocytosine in laboratory mutants of Candida glabrata.

Resistance to 5-fluorocytosine (5-FC) has been poorly investigated in the yeast Candida glabrata. This study was conducted on laboratory mutants obtained by exposure of a wild-type isolate to 5-FC. Based on their susceptibility to 5-fluorouracil (5-FU), two of these mutants were selected for further analysis of the molecular mechanisms of 5-FC resistance. One mutant, resistant to both compounds, exhibited a missense mutation in the gene coding the cytosine deaminase and a decrease in the expression level of the gene coding the uridine monophosphate pyrophosphorylase. The other mutant that showed a reduced susceptibility to 5-FC and 5-FU exhibited an overexpression of the genes coding the thymidylate synthase and a cytosine permease, associated with a missense mutation in the last gene. Thus, beside mutations in the FUR1 gene which represent the most common cause of resistance to 5-FC, other mechanisms may also occur in C. glabrata.