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Patients with colorectal liver metastasis (CLM) present with heterogenous clinical outcomes and improved classification is needed to ameliorate the therapeutic output. Macrophages (Mϕ) hold promise as prognostic classifiers and therapeutic targets. Here, stemming from a single-cell analysis of mononuclear phagocytes infiltrating human CLM, we identified two Mϕ markers associated with distinct populations with opposite clinical relevance. The invasive margin of CLM was enriched in pro-inflammatory monocyte-derived Mϕ (MoMϕ) expressing the monocytic marker SERPINB2, and a more differentiated population, tumor-associated Mϕ (TAM), expressing glycoprotein nonmetastatic melanoma protein B (GPNMB). SERPINB2+ MoMϕ had an early inflammatory profile, whereas GPNMB+ TAMs were enriched in pathways of matrix degradation, angiogenesis, and lipid metabolism and were found closer to the tumor margin, as confirmed by spatial transcriptomics on CLM specimens. In a cohort of patients, a high infiltration of SERPINB2+ cells independently associated with longer disease-free survival (DFS; P = 0.033), whereas a high density of GPNMB+ cells correlated with shorter DFS (P = 0.012) and overall survival (P = 0.002). Cell–cell interaction analysis defined opposing roles for MoMϕ and TAMs, suggesting that SERPINB2+ and GPNMB+ cells are discrete populations of Mϕ and may be exploited for further translation to an immune-based stratification tool. This study provides evidence of how multi-omics approaches can identify nonredundant, clinically relevant markers for further translation to immune-based patient stratification tools and therapeutic targets. GPNMB has been shown to set Mϕ in an immunosuppressive mode. Our high dimensional analyses provide further evidence that GPNMB is a negative prognostic indicator and a potential player in the protumor function of Mϕ populations.

The landscape and function of the immune infiltrate of intrahepatic cholangiocarcinoma (iCCA), a rare, yet aggressive tumor of the biliary tract, remains poorly characterized, limiting development of successful immunotherapies. Herein, we aimed to define the molecular characteristics of tumor-infiltrating leukocytes with a special focus on CD4+ regulatory T cells (Tregs).We used high-dimensional single-cell technologies to characterize the T-cell and myeloid compartments of iCCA tissues, comparing these with their tumor-free peritumoral and circulating counterparts. We further used genomics and cellular assays to define the iCCA-specific role of a novel transcription factor, mesenchyme homeobox 1 (MEOX1), in Treg biology.We found poor infiltration of putative tumor-specific CD39+ CD8+ T cells accompanied by abundant infiltration of hyperactivated CD4+ Tregs. Single-cell RNA-sequencing identified an altered network of transcription factors in iCCA-infiltrating compared to peritumoral T cells, suggesting reduced effector functions by tumor-infiltrating CD8+ T cells and enhanced immunosuppression by CD4+ Tregs. Specifically, we found that expression of MEOX1 was highly enriched in tumor-infiltrating Tregs, and demonstrated that MEOX1 overexpression is sufficient to reprogram circulating Tregs to acquire the transcriptional and epigenetic landscape of tumor-infiltrating Tregs. Accordingly, enrichment of the MEOX1-dependent gene program in Tregs was strongly associated with poor prognosis in a large cohort of patients with iCCA.We observed abundant infiltration of hyperactivated CD4+ Tregs in iCCA tumors along with reduced CD8+ T-cell effector functions. Interfering with hyperactivated Tregs should be explored as an approach to enhance antitumor immunity in iCCA.Immune cells have the potential to slow or halt the progression of tumors. However, some tumors, such as intrahepatic cholangiocarcinoma, are associated with very limited immune responses (and infiltration of cancer-targeting immune cells). Herein, we show that a specific population of regulatory T cells (a type of immune cell that actually suppresses the immune response) are hyperactivated in intrahepatic cholangiocarcinoma. Targeting these cells could enable cancer-targeting immune cells to act more effectively and should be looked at as a potential therapeutic approach to this aggressive cancer type.

Mitofusin-2 (MFN2) is an outer mitochondrial membrane protein essential for mitochondrial networking in most cells. Autosomal dominant mutations in the MFN2 gene cause Charcot-Marie-Tooth type 2A disease (CMT2A), a severe and disabling sensory-motor neuropathy that impacts the entire nervous system. Here, we propose a novel therapeutic strategy tailored to correcting the root genetic defect of CMT2A. Though mutant and wild-type MFN2 mRNA are inhibited by RNA interference (RNAi), the wild-type protein is restored by overexpressing cDNA encoding functional MFN2 modified to be resistant to RNAi. We tested this strategy in CMT2A patient-specific human induced pluripotent stem cell (iPSC)-differentiated motor neurons (MNs), demonstrating the correct silencing of endogenous MFN2 and replacement with an exogenous copy of the functional wild-type gene. This approach significantly rescues the CMT2A MN phenotype in vitro, stabilizing the altered axonal mitochondrial distribution and correcting abnormal mitophagic processes. The MFN2molecular correction was also properly confirmed in vivo in the MitoCharc1 CMT2A transgenic mouse model after cerebrospinal fluid (CSF) delivery of the constructs into newborn mice using adeno-associated virus 9 (AAV9). Altogether, our data support the feasibility of a combined RNAi and gene therapy strategy for treating the broad spectrum of human diseases associated with MFN2mutations.

Supplementary Figures 1-4, Tables S1 and S5

Supplementary Table S4

Patients with colorectal liver metastasis (CLM) present with heterogenous clinical outcomes and improved classification is needed to ameliorate the therapeutic output. Macrophages (Mϕ) hold promise as prognostic classifiers and therapeutic targets. Here, stemming from a single-cell analysis of mononuclear phagocytes infiltrating human CLM, we identified two Mϕ markers associated with distinct populations with opposite clinical relevance. The invasive margin of CLM was enriched in pro-inflammatory monocyte-derived Mϕ (MoMϕ) expressing the monocytic marker SERPINB2, and a more differentiated population, tumor-associated Mϕ (TAM), expressing glycoprotein nonmetastatic melanoma protein B (GPNMB). SERPINB2+ MoMϕ had an early inflammatory profile, whereas GPNMB+ TAMs were enriched in pathways of matrix degradation, angiogenesis, and lipid metabolism and were found closer to the tumor margin, as confirmed by spatial transcriptomics on CLM specimens. In a cohort of patients, a high infiltration of SERPINB2+ cells independently associated with longer disease-free survival (DFS; P = 0.033), whereas a high density of GPNMB+ cells correlated with shorter DFS (P = 0.012) and overall survival (P = 0.002). Cell–cell interaction analysis defined opposing roles for MoMϕ and TAMs, suggesting that SERPINB2+ and GPNMB+ cells are discrete populations of Mϕ and may be exploited for further translation to an immune-based stratification tool. This study provides evidence of how multi-omics approaches can identify nonredundant, clinically relevant markers for further translation to immune-based patient stratification tools and therapeutic targets. GPNMB has been shown to set Mϕ in an immunosuppressive mode. Our high dimensional analyses provide further evidence that GPNMB is a negative prognostic indicator and a potential player in the protumor function of Mϕ populations.

Supplementary figure legends

Aims Heart failure with reduced ejection fraction (HFrEF) is a leading cause of mortality worldwide, requiring novel therapeutic and lifestyle interventions. Metabolic alterations and energy production deficit are hallmarks and thereby promising therapeutic targets for this complex clinical syndrome. We aim to study the molecular mechanisms and effects on cardiac function in rodents with HFrEF of a designer diet in which free essential amino acids—in specifically designed percentages—substituted for protein. Methods and results Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricle (LV) pressure overload or sham surgery. Whole-body glucose homeostasis was studied with glucose tolerance test, while myocardial dysfunction and fibrosis were measured with echocardiogram and histological analysis. Mitochondrial bioenergetics and morphology were investigated with oxygen consumption rate measurement and electron microscopy evaluation. Circulating and cardiac non-targeted metabolite profiles were analyzed by ultrahigh performance liquid chromatography-tandem mass spectroscopy, while RNA-sequencing was used to identify signalling pathways mainly affected. The amino acid-substituted diet shows remarkable preventive and therapeutic effects. This dietary approach corrects the whole-body glucose metabolism and restores the unbalanced metabolic substrate usage—by improving mitochondrial fuel oxidation—in the failing heart. In particular, biochemical, molecular, and genetic approaches suggest that renormalization of branched-chain amino acid oxidation in cardiac tissue, which is suppressed in HFrEF, plays a relevant role. Beyond the changes of systemic metabolism, cell-autonomous processes may explain at least in part the diet’s cardioprotective impact. Conclusion Collectively, these results suggest that manipulation of dietary amino acids, and especially essential amino acids, is a potential adjuvant therapeutic strategy to treat systolic dysfunction and HFrEF in humans.

Background: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation’s potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. Methods: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf -deficient ( Mydgf −/− ), and Mydgf bone marrow–chimeric or bone marrow–conditional transgenic mice with pressure overload–induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography–mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca 2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. Results: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf −/− mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow–derived inflammatory cells attenuated pressure overload–induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein–coupled receptor agonist–induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca 2+ -ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf −/− mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca 2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf −/− mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload–induced hypertrophy and dysfunction. Pressure-overloaded Mydgf −/− mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. Conclusions: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload–induced heart failure.

Mechanisms by which specific histone modifications regulate distinct gene regulatory networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression in mammalian cardiogenesis. Early embryonic cardiomyocyte ablation ofDot1lrevealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific gene regulatory networks at two critical cardiogenic junctures: left ventricle patterning and postnatal cardiomyocyte cell cycle withdrawal. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, these analyses also revealed that H3K79me2 in specific regulatory elements contributed to silencing genes usually not expressed in cardiomyocytes. As DOT1L mutants had increased numbers of postnatal mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity, controlled inhibition of DOT1L might be a strategy to promote cardiac regeneration post-injury.

More than 50% of all patients with colorectal cancer (CRC) develop liver metastases (CLM), a clinical condition characterized by poor prognosis and lack of reliable prognostic markers. Vδ1 cells are a subset of tissue-resident gamma delta (γδ) T lymphocytes endowed with a broad array of antitumor functions and showing a natural high tropism for the liver. However, little is known about their impact in the clinical outcomes of CLM.We isolated human γδ T cells from peripheral blood (PB) and peritumoral (PT) tissue of 93 patients undergone surgical procedures to remove CLM. The phenotype of freshly purified γδ T cells was assessed by multiparametric flow cytometry, the transcriptional profiles by single cell RNA-sequencing, the functional annotations by Gene Ontology enrichment analyses and the clonotype by γδ T cell receptor (TCR)-sequencing.The microenvironment of CLM is characterized by a heterogeneous immune infiltrate comprising different subsets of γδ tumor-infiltrating lymphocytes (TILs) able to egress the liver and re-circulate in PB. Vδ1 T cells represent the largest population of γδ TILs within the PT compartment of CLM that is greatly enriched in Vδ1 T effector (TThe enrichment of tissue-resident CD69

Introduction: The ongoing coronavirus disease 19 (COVID-19) outbreak involves the pediatric population, but to date, few reports have investigated the circulation of variants among children. Material and Methods: In this retrospective study, non-hospitalized pediatric patients with SARS-CoV-2-positive nasopharyngeal swabs (NPS) were enrolled at the Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste (Italy), from November 2020 to January 2022. SARS-CoV-2 variants were identified by in vitro viral isolation, amplification, automatic sequencing of the receptor binding domain (RBD) of the SARS-CoV-2 spike coding gene, and subsequent next-generation sequencing. The growth curves of the isolated strains were defined in vitro by infecting Vero-E6 cells and quantifying the viral load in the supernatants up to 72 h post-infection by qRT–PCR. The neutralization activity of sera obtained from a COVID-19 vaccinated subject, recovered (2020) patient, vaccinated and recovered (2021) patient, and seronegative subject was assessed by microneutralization assay against the different variants. Results: In total, 32 SARS-CoV-2-positive children, 16 (50%) females, with a median age of 1.4 years (range: 1 day–13 years), were enrolled. The D614G amino acid substitution was detected in all isolated and amplified viral strains. Of the 32 isolates, 4 (12.5%) carried a nonsynonymous nucleotide mutation leading to the N439K (3/4), lineage B.1.258 (∆H69/∆V70), and S477N (1/4) substitution. In 7/32 (21.8%) isolates, amino acid substitutions allowed the identification of a delta variant, lineage B.1.617.2-AY.43, and in 1/32 (3.1%), the Omicron strain (B.1.1.529.BA1) was identified. The growth curves of the B.1, B.1.258 (∆H69/∆V70), B.1.617.2-AY.43, and B.1.1.529.BA1 variants did not show any significant differences. A reduction in the serum neutralizing activity against B.1.258 (∆H69/∆V70) only in a vaccinated subject (1.7-fold difference), against B.1.617.2-AY.43 in a vaccinated subject and in recovered patients (12.7 and ≥2.5-fold differences, respectively), and against B.1.1.529.BA1 variant (57.6- and 1.4-fold differences in vaccinated and in vaccinated and recovered patients) were observed compared to the B.1 variant. Conclusions: SARS-CoV-2 variants carrying the B.1.258 (∆H69/∆V70) and S477N substitutions were reported here in a pediatric population for the first time. Although the growth rates of the isolated strains (B.1.258, B.1.617.2-AY.43, B.1.1.529.BA1) did not differ from the B.1 variant, neutralizing activity of the sera from vaccinated subjects significantly decreased against these variants. Attention should be devoted to the pediatric population to prevent the spread of new SARS-CoV-2 variants in an unvaccinated and predominantly naive population.

Introduction: The ongoing coronavirus disease 19 (COVID-19) outbreak involves the pediatric population, but to date, few reports have investigated the circulation of variants among children. Material and Methods: In this retrospective study, non-hospitalized pediatric patients with SARS-CoV-2-positive nasopharyngeal swabs (NPS) were enrolled at the Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste (Italy), from November 2020 to January 2022. SARS-CoV-2 variants were identified by in vitro viral isolation, amplification, automatic sequencing of the receptor binding domain (RBD) of the SARS-CoV-2 spike coding gene, and subsequent nextgeneration sequencing. The growth curves of the isolated strains were defined in vitro by infecting Vero-E6 cells and quantifying the viral load in the supernatants up to 72 h post-infection by qRT– PCR. The neutralization activity of sera obtained from a COVID-19 vaccinated subject, recovered (2020) patient, vaccinated and recovered (2021) patient, and seronegative subject was assessed by microneutralization assay against the different variants. Results: In total, 32 SARS-CoV-2-positive children, 16 (50%) females, with a median age of 1.4 years (range: 1 day–13 years), were enrolled. The D614G amino acid substitution was detected in all isolated and amplified viral strains. Of the 32 isolates, 4 (12.5%) carried a nonsynonymous nucleotide mutation leading to the N439K (3/4), lineage B.1.258 (∆H69/∆V70), and S477N (1/4) substitution. In 7/32 (21.8%) isolates, amino acid substitutions allowed the identification of a delta variant, lineage B.1.617.2-AY.43, and in 1/32 (3.1%), the Omicron strain (B.1.1.529.BA1) was identified. The growth curves of the B.1, B.1.258 (∆H69/∆V70), B.1.617.2-AY.43, and B.1.1.529.BA1 variants did not show any significant differences. A reduction in the serum neutralizing activity against B.1.258 (∆H69/∆V70) only in a vaccinated subject (1.7-fold difference), against B.1.617.2-AY.43 in a vaccinated subject and in recovered patients (12.7 and ≥2.5-fold differences, respectively), and against B.1.1.529.BA1 variant (57.6-and 1.4-fold differences in vaccinated and in vaccinated and recovered patients) were observed compared to the B.1 variant. Conclusions: SARS-CoV-2 variants carrying the B.1.258 (∆H69/∆V70) and S477N substitutions were reported here in a pediatric population for the first time. Although the growth rates of the isolated strains (B.1.258, B.1.617.2-AY.43, B.1.1.529.BA1) did not differ from the B.1 variant, neutralizing activity of the sera from vaccinated subjects significantly decreased against these variants. Attention should be devoted to the pediatric population to prevent the spread of new SARS-CoV-2 variants in an unvaccinated and predominantly naive population.

Tumor-associated macrophages (TAMs) are correlated with the progression of prostatic adenocarcinoma (PCa). The mechanistic basis of this correlation and therapeutic strategies to target TAMs in PCa remain poorly defined. Here, single-cell RNA sequencing was used to profile the transcriptional landscape of TAMs in human PCa, leading to identification of a subset of macrophages characterized by dysregulation in transcriptional pathways associated with lipid metabolism. This subset of TAMs correlates positively with PCa progression and shorter disease-free survival and is characterized by an accumulation of lipids that is dependent on Marco. Mechanistically, cancer cell–derived IL-1β enhances Marco expression on macrophages, and reciprocally, cancer cell migration is promoted by CCL6 released by lipid-loaded TAMs. Moreover, administration of a high-fat diet to tumor-bearing mice raises the abundance of lipid-loaded TAMs. Finally, targeting lipid accumulation by Marco blockade hinders tumor growth and invasiveness and improves the efficacy of chemotherapy in models of PCa, pointing to combinatorial strategies that may influence patient outcomes.

Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation's potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload.We defined the cellular sources and function of MYDGF in wild-type (WT),MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. WhileThese findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload-induced heart failure.

The TMPRSS2-ERG gene fusion is the most frequent alteration observed in human prostate cancer. However, its role in disease progression is still unclear. In this study, we uncover an important mechanism promoting ERG oncogenic activity. We show that ERG is methylated by Enhancer of zest homolog 2 (EZH2) at a specific lysine residue (K362) located within the internal auto-inhibitory domain. Mechanistically, K362 methylation modifies intra-domain interactions, favors DNA binding and enhances ERG transcriptional activity. In a genetically engineered mouse model of ERG fusion-positive prostate cancer (Pb-Cre4 Pten flox/flox Rosa26-ERG, ERG/PTEN), ERG K362 methylation is associated with PTEN loss and progression to invasive adenocarcinomas. In both ERG positive VCaP cells and ERG/PTEN mice, PTEN loss results in AKT activation and EZH2 phosphorylation at serine 21 that favors ERG methylation. We find that ERG and EZH2 interact and co-occupy several sites in the genome forming trans-activating complexes. Consistently, ERG/EZH2 co-regulated target genes are deregulated preferentially in tumors with concomitant ERG gain and PTEN loss and in castration-resistant prostate cancers. Collectively, these findings identify ERG methylation as a post-translational modification sustaining disease progression in ERG-positive prostate cancers., Although the TMPRSS2-ERG gene fusion is the most common alteration in human prostate cancer, its involvement in disease progression remains unclear. Here, the authors demonstrate that ERG is methylated by Enhancer of zest homolog 2 leading to enhanced transcriptional and oncogenic activity.

Background: Inflammation is a key component of cardiac disease, with macrophages and T lymphocytes mediating essential roles in the progression to heart failure. Nonetheless, little insight exists on other immune subsets involved in the cardiotoxic response. Methods: Here, we used single-cell RNA sequencing to map the cardiac immune composition in the standard murine nonischemic, pressure-overload heart failure model. By focusing our analysis on CD45 + cells, we obtained a higher resolution identification of the immune cell subsets in the heart, at early and late stages of disease and in controls. We then integrated our findings using multiparameter flow cytometry, immunohistochemistry, and tissue clarification immunofluorescence in mouse and human. Results: We found that most major immune cell subpopulations, including macrophages, B cells, T cells and regulatory T cells, dendritic cells, Natural Killer cells, neutrophils, and mast cells are present in both healthy and diseased hearts. Most cell subsets are found within the myocardium, whereas mast cells are found also in the epicardium. Upon induction of pressure overload, immune activation occurs across the entire range of immune cell types. Activation led to upregulation of key subset-specific molecules, such as oncostatin M in proinflammatory macrophages and PD-1 in regulatory T cells, that may help explain clinical findings such as the refractivity of patients with heart failure to anti–tumor necrosis factor therapy and cardiac toxicity during anti–PD-1 cancer immunotherapy, respectively. Conclusions: Despite the absence of infectious agents or an autoimmune trigger, induction of disease leads to immune activation that involves far more cell types than previously thought, including neutrophils, B cells, Natural Killer cells, and mast cells. This opens up the field of cardioimmunology to further investigation by using toolkits that have already been developed to study the aforementioned immune subsets. The subset-specific molecules that mediate their activation may thus become useful targets for the diagnostics or therapy of heart failure.

Description, The inner workings of the clock system rely on communicating signals between distal tissues to maintain daily metabolism., The mammalian circadian clock, expressed throughout the brain and body, controls daily metabolic homeostasis. Clock function in peripheral tissues is required, but not sufficient, for this task. Because of the lack of specialized animal models, it is unclear how tissue clocks interact with extrinsic signals to drive molecular oscillations. Here, we isolated the interaction between feeding and the liver clock by reconstituting Bmal1 exclusively in hepatocytes (Liver-RE), in otherwise clock-less mice, and controlling timing of food intake. We found that the cooperative action of BMAL1 and the transcription factor CEBPB regulates daily liver metabolic transcriptional programs. Functionally, the liver clock and feeding rhythm are sufficient to drive temporal carbohydrate homeostasis. By contrast, liver rhythms tied to redox and lipid metabolism required communication with the skeletal muscle clock, demonstrating peripheral clock cross-talk. Our results highlight how the inner workings of the clock system rely on communicating signals to maintain daily metabolism.

SummaryEarly prenatal inflammatory conditions are thought to represent a risk factor for different neurodevelopmental disorders, with long-term consequences on adult brain connectivity. Here we show that a transient IL-6 elevation, occurring at vulnerable stages of early neurodevelopment, directly impacts brain developmental trajectories through the aberrant enhancement of glutamatergic synapses and overall brain hyper-connectivity. The IL6-mediated boost of excitatory synapse density results from the neuron-autonomous, genomic effect of the transcription factor STAT3 and causally involves the activation of RGS4 gene as a candidate downstream target. The STAT3/RGS4 pathway is also activated in neonatal brains as a consequence of maternal immune activation protocols mimicking a viral infection during pregnancy. By demonstrating that prenatal IL-6 elevations result in aberrant synaptic and brain connectivity through the molecular players identified, we provide a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

Early prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

Although the pathological significance of tumor-associated macrophage (TAM) heterogeneity is still poorly understood, TAM reprogramming is viewed as a promising anticancer therapy. Here we show that a distinct subset of TAMs (F4/80hiCD115hiC3aRhiCD88hi), endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), plays a critical role in shaping a prometastatic tumor microenvironment favoring immunosuppression, angiogenesis and epithelial-to-mesenchymal transition. This population originates from F4/80+HO-1+ bone marrow (BM) precursors, accumulates in the blood of tumor bearers and preferentially localizes at the invasive margin through a mechanism dependent on the activation of Nrf2 and coordinated by the NF-κB1-CSF1R-C3aR axis. Inhibition of F4/80+HO-1+ TAM recruitment or myeloid-specific deletion of HO-1 blocks metastasis formation and improves anticancer immunotherapy. Relative expression of HO-1 in peripheral monocyte subsets, as well as in tumor lesions, discriminates survival among metastatic melanoma patients. Overall, these results identify a distinct cancer-induced HO-1+ myeloid subgroup as a new antimetastatic target and prognostic blood marker.

Summary Enhancer RNAs (eRNAs) are a subset of long noncoding RNA generated from genomic enhancers: they are thought to act as potent promoters of the expression of nearby genes through interaction with the transcriptional and epigenomic machineries. In the present work, we describe two eRNAs transcribed from the enhancer of Nkx2-5—a gene specifying a master cardiomyogenic lineage transcription factor (TF)—which we call Intergenic Regulatory Element Nkx2-5 Enhancers (IRENEs). The IRENEs are encoded, respectively, on the same strand (SS) and in the divergent direction (div) respect to the nearby gene. Of note, these two eRNAs have opposing roles in the regulation of Nkx2-5: IRENE-SS acts as a canonical promoter of transcription, whereas IRENE-div represses the activity of the enhancer through recruitment of the histone deacetylase sirtuin 1. Thus, we have identified an autoregulatory loop controlling expression of the master cardiac TF NKX2-5, in which one eRNA represses transcription., Graphical Abstract, Highlights • Two eRNAs (IRENE-SS, IRENE-div) with opposing functions are found upstream of Nkx2-5 • IRENE-SS works as a classical eRNA, acting as a transcriptional activator • IRENE-div acts unconventionally, functioning as a transcriptional repressor • IRENEs epigenetically control enhancer status and, subsequently, locus architecture, Biological Sciences; Molecular Biology; Molecular Mechanism of Gene Regulation

CRISPR/Cas9 has become a powerful method to engineer genomes and to activate or to repress genes expression. As such, in cancer research CRISPR/Cas9 technology represents an efficient tool to dissect mechanisms of tumorigenesis and to discover novel targets for drug development. Here, we employed the CRISPR/Cas9 technology for studying the role of prostaglandin-endoperoxide synthase 2 (PTGS2) in melanoma development and progression. Melanoma is the most aggressive form of skin cancer with a median survival of less than 1 year. Although oncogene-targeted drugs and immune checkpoint inhibitors have demonstrated a significant success in improving overall survival in patients, related toxicity and emerging resistance are ongoing challenges. Gene therapy appears to be an appealing option to enhance the efficacy of currently available melanoma therapeutics leading to better patient prognosis. Several gene therapy targets have been identified and have proven to be effective against melanoma cells. Particularly, PTGS2 is frequently expressed in malignant melanomas and its expression significantly correlates with poor survival in patients. In this study we investigated on the effect of ptgs2 knockdown in B16F10 murine melanoma cells. Our results show that reduced expression of ptgs2 in melanoma cells: i) inhibits cell proliferation, migration, and invasiveness; ii) modulates immune response by impairing myeloid derived suppressor cell differentiation; iii) reduces tumor development and metastasis in vivo. Collectively, these findings indicate that ptgs2 could represent an ideal gene to be targeted to improve success rates in the development of new and highly selective drugs for melanoma treatment.

Background The role of microRNA as biomarkers able to predict major coronary events (MACE) has not been fully elucidated, reproducibility being a critical issue. Aim To identify circulating microRNA signatures able to predict MACE. Methods We employed a PCR-based method to screen 754 microRNAs in a cohort of 60-year-olds (60YOs) from Stockholm, using a nested case-control design (100 cases vs 100 matched controls). The association of microRNAs and their interaction with the risk of MACE (myocardial infarction (MI), angina and sudden cardiac death) was estimated with random-effect logistic regression and expressed as OR with 95% CI. A bioinformatics approach identified microRNA clusters based on predicted targets. Main findings were tested in 58 MI and 60 age and sex matched referents from the the Nord-Trøndelag Health (HUNT) Study, a longitudinal population health study conducted in Norway. Results Fifty-five microRNAs were found to be associated with risk of MACE in the 60YO. MicroRNA-145-3p was associated with the largest estimated risk increase of MACE after adjustment for the common CV risk factors (OR: 2.18; 95% CI: 1.27–3.75). Interaction analysis revealed that increasing plasma levels of microRNA-320b modulated the association of 16 microRNAs with risk of MACE. As an example the estimated MACE risk associated with microRNA-145-3p was 1.47 (0.87–2.47) in the presence of low (75th percentile) miRNA 320b expression levels. Sixteen microRNA pairs could be classified in 4 functional clusters with 492 predicted gene targets, mainly involved in the regulation of inflammation, thrombosis and lipid metabolism. Eight miRNAs interacting pairs belonging to cluster 2 and 4 showed a similar association trend with MI risk in the HUNT study. Conclusions We report the identification of microRNA signatures predicting risk of MACE in middle-aged Scandinavian men and women. These signatures could be a valuable tool to improve CV disease prediction in the aged.

Macrophage morphology could be used to stratify metastasis risk for CRC patients., Using macrophage morphology in human colorectal cancer liver metastasis, Donadon et al. in this issue of JEM (https://doi.org/10.1084/jem.20191847) provide a window into lipid metabolism and foamy macrophages, which accrue in numerous pathological states and here are shown to have clinical application.

Adult vascular smooth muscle cells (VSMCs) dedifferentiate in response to extracellular cues such as vascular damage and inflammation. Dedifferentiated VSMCs are proliferative, migratory, less contractile, and can contribute to vascular repair as well as to cardiovascular pathologies such as intimal hyperplasia/restenosis in coronary artery and arterial aneurysm. We here demonstrate the role of ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) as an epigenetic master regulator of VSMC plasticity. UHRF1 expression correlated with the development of vascular pathologies associated with modulation of noncoding RNAs, such as microRNAs. miR-145 — pivotal in regulating VSMC plasticity, which is reduced in vascular diseases — was found to control Uhrf1 mRNA translation. In turn, UHRF1 triggered VSMC proliferation, directly repressing promoters of cell-cycle inhibitor genes (including p21 and p27) and key prodifferentiation genes via the methylation of DNA and histones. Local vascular viral delivery of Uhrf1 shRNAs or Uhrf1 VSMC-specific deletion prevented intimal hyperplasia in mouse carotid artery and decreased vessel damage in a mouse model of aortic aneurysm. Our study demonstrates the fundamental role of Uhrf1 in regulating VSMC phenotype by promoting proliferation and dedifferentiation. UHRF1 targeting may hold therapeutic potential in vascular pathologies.

Mutations in LMNA, which encodes the nuclear proteins Lamin A/C, can cause cardiomyopathy and conduction disorders. Here, we employ induced pluripotent stem cells (iPSCs) generated from human cells carrying heterozygous K219T mutation on LMNA to develop a disease model. Cardiomyocytes differentiated from these iPSCs, and which thus carry K219T-LMNA, have altered action potential, reduced peak sodium current and diminished conduction velocity. Moreover, they have significantly downregulated Nav1.5 channel expression and increased binding of Lamin A/C to the promoter of SCN5A, the channel’s gene. Coherently, binding of the Polycomb Repressive Complex 2 (PRC2) protein SUZ12 and deposition of the repressive histone mark H3K27me3 are increased at SCN5A. CRISPR/Cas9-mediated correction of the mutation re-establishes sodium current density and SCN5A expression. Thus, K219T-LMNA cooperates with PRC2 in downregulating SCN5A, leading to decreased sodium current density and slower conduction velocity. This mechanism may underlie the conduction abnormalities associated with LMNA-cardiomyopathy., Mutation of LMNA, encoding Lamin A/C nuclear proteins, cause dilated cardiomyopathy and conduction disorders. Here, the authors show that patient-specific iPSC-derived CMs carrying the K219T LMNA mutation have downregulated Nav1.5 channels due to dynamic cooperation of Lamin A/C and Polycomb repressor complex 2 at the SCN5A promoter.

Summary Neutrophils are a component of the tumor microenvironment and have been predominantly associated with cancer progression. Using a genetic approach complemented by adoptive transfer, we found that neutrophils are essential for resistance against primary 3-methylcholantrene-induced carcinogenesis. Neutrophils were essential for the activation of an interferon-γ-dependent pathway of immune resistance, associated with polarization of a subset of CD4− CD8− unconventional αβ T cells (UTCαβ). Bulk and single-cell RNA sequencing (scRNA-seq) analyses unveiled the innate-like features and diversity of UTCαβ associated with neutrophil-dependent anti-sarcoma immunity. In selected human tumors, including undifferentiated pleomorphic sarcoma, CSF3R expression, a neutrophil signature and neutrophil infiltration were associated with a type 1 immune response and better clinical outcome. Thus, neutrophils driving UTCαβ polarization and type 1 immunity are essential for resistance against murine sarcomas and selected human tumors., Graphical Abstract, Highlights • Neutrophils mediate antitumor response by sustaining an IL-12/IFNγ-dependent pathway • Neutrophils are essential for unconventional αβ T cell (UTCαβ) type 1 polarization • Type 1 UTCαβ possess an innate-like phenotype and display antitumor potential in vivo • Neutrophil infiltration is associated with good prognosis in selected human tumors, Tumor-associated neutrophils (TANs) have mainly been portrayed as tumor-promoters. Here, we describe a novel antitumor pathway in which TANs promote IL-12 production by macrophages, leading to type 1 polarization of a subset of unconventional αβ T cell (UTCαβ). Type 1 UTCαβ possess an innate-like phenotype and antitumor potential in vivo. In selected human tumors, neutrophil infiltration is associated with type 1 immunity and better clinical outcome.

Mesenchymal stem cells (MSCs) are well established to have promising therapeutic properties. TNF-stimulated gene-6 (TSG-6), a potent tissue-protective and anti-inflammatory factor, has been demonstrated to be responsible for a significant part of the tissue-protecting properties mediated by MSCs. Nevertheless, current knowledge about the biological function of TSG-6 in MSCs is limited. Here, we demonstrated that TSG-6 is a crucial factor that influences many functional properties of MSCs. The transcriptomic sequencing analysis of wild-type (WT) and TSG-6−/−-MSCs shows that the loss of TSG-6 expression leads to the perturbation of several transcription factors, cytokines, and other key biological pathways. TSG-6−/−-MSCs appeared morphologically different with dissimilar cytoskeleton organization, significantly reduced size of extracellular vesicles, decreased cell proliferative rate, and loss of differentiation abilities compared with the WT cells. These cellular effects may be due to TSG-6-mediated changes in the extracellular matrix (ECM) environment. The supplementation of ECM with exogenous TSG-6, in fact, rescued cell proliferation and changes in morphology. Importantly, TSG-6-deficient MSCs displayed an increased capacity to release interleukin-6 conferring pro-inflammatory and pro-tumorigenic properties to the MSCs. Overall, our data provide strong evidence that TSG-6 is crucial for the maintenance of stemness and other biological properties of murine MSCs.