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Neutrophils are the first immune cells to kill invading microbes at sites of infection using a variety of processes,including the release of proteases,phagocytosis and the production of neutrophil extracellular traps (NETs). NET formation,or NETosis,is a specific and highly efficient process,which is induced by a variety of stimuli leading to expulsion of DNA,proteases and antimicrobial peptides to the extracellular space. However,uncontrolled NETosis may lead to adverse effects and exert tissue damage in pathological conditions. Here,we show that the ATP channel pannexin1 (Panx1) is functionally expressed by bone marrow-derived neutrophils (BMDNs) of wild-type (WT) mice and that ATP contributes to NETosis induced in vitro by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate (PMA). Interestingly,neutrophils isolated from Panx1-/- mice showed reduced and/or delayed induction of NETosis. Brilliant blue FCF dye (BB-FCF),a Panx1 channel inhibitor,decreased NETosis in wild-type neutrophils to the extent observed in Panx1-/- neutrophils. Thus,we demonstrate that ATP and Panx1 channels contribute to NETosis and may represent a therapeutic target. View Publication

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age,but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further,we examined the combined and separate effects of an oncogene (c-MYC) and exposure to IL3,GM-CSF and SCF on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized,rapidly fatal and serially transplantable blast population,phenotypically and transcriptionally similar to human AML cells,but only in mice producing IL3,GM-CSF and SCF transgenically,or in regular mice in which the cells were exposed to IL3 or GM-CSF delivered using a co-transduction strategy. In their absence,the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months but,upon transfer to secondary mice producing the human cytokines,the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38-) and late (GMPs) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them. View Publication

Tissue resident intestinal macrophages are known to exhibit an anti-inflammatory phenotype and produce little pro-inflammatory cytokines upon TLR ligation,allowing symbiotic co-existence with the intestinal microbiota. However,upon acute events such as epithelial damage and concomitant influx of microbes,these macrophages must be able to quickly mount a pro-inflammatory response while more inflammatory macrophages are recruited from the blood stream simultaneously. Here,we show that dietary intake of vitamin A is required for the maintenance of the anti-inflammatory state of tissue resident intestinal macrophages. Interestingly,these anti-inflammatory macrophages were characterized by high levels of Dectin-1 expression. We show that Dectin-1 expression is enhanced by the vitamin A metabolite retinoic acid and our data suggests that Dectin-1 triggering might provide a switch to induce a rapid production of pro-inflammatory cytokines. In addition,Dectin-1 stimulation resulted in an altered metabolic profile which is linked to a pro-inflammatory response. Together,our data suggests that presence of vitamin A in the small intestine enhances an anti-inflammatory phenotype as well as Dectin-1 expression by macrophages and that this anti-inflammatory phenotype can rapidly convert toward a pro-inflammatory state upon Dectin-1 signaling. View Publication

Mannose is a glucose-associated serum metabolite mainly released by the liver. Recent studies have shown several unexpected pleiotropic effects of mannose including increased regulatory T cells (Tregs),prevention of auto-immune disease and ability to reduce growth of human cancer cells. We have previously shown in large cohorts that elevated serum mannose levels are associated with future development of type 2 diabetes (T2D) and cardiovascular disease. However,potential direct effects of mannose on insulin sensitivity in vivo or in vitro are unknown. We here show that administration of mannose (0.1 g/kg BW twice daily) for one week in man did not elicit negative effects on meal-modified glucose tolerance,markers of inflammation or insulin levels. Tregs number and insulin signaling in human liver cells were unchanged. These data suggest that mannose is a marker,and not a mediator,of insulin resistance. To verify this,we examined serum mannose levels during long-term euglycemic hyperinsulinemic clamps in non-diabetic and T2D individuals. Mannose was reduced by insulin infusion in proportion to whole-body insulin sensitivity. Thus,mannose is a biomarker of insulin resistance which may be useful for the early identification of diabetic individuals with insulin resistance and increased risk of its complications. View Publication

Activation-induced cytidine deaminase (AID) mediates immunoglobulin class switch DNA recombination (CSR) and somatic hypermutation (SHM),critical processes for maturation of the antibody response. Epigenetic factors,such as histone deacetylases (HDACs),would underpin B cell differentiation stage-specific AID expression. Here,we showed that NAD+-dependent class III HDAC sirtuin 1 (Sirt1) is highly expressed in resting B cells and down-regulated by stimuli inducing AID. B cell Sirt1 down-regulation,deprivation of NAD+ cofactor,or genetic Sirt1 deletion reduced deacetylation of Aicda promoter histones,Dnmt1,and nuclear factor-$\kappa$B (NF-$\kappa$B) p65 and increased AID expression. This promoted class-switched and hypermutated T-dependent and T-independent antibody responses or led to generation of autoantibodies. Genetic Sirt1 overexpression,Sirt1 boost by NAD+,or allosteric Sirt1 enhancement by SRT1720 repressed AID expression and CSR/SHM. By deacetylating histone and nonhistone proteins (Dnmt1 and NF-$\kappa$B p65),Sirt1 transduces metabolic cues into epigenetic changes to play an important B cell-intrinsic role in modulating antibody and autoantibody responses. View Publication

Transcriptional status determines the HIV replicative state in infected patients. However,the transcriptional mechanisms for proviral replication control remain unclear. In this study,we show that,apart from its function in HIV integration,LEDGF/p75 differentially regulates HIV transcription in latency and proviral reactivation. During latency,LEDGF/p75 suppresses proviral transcription via promoter-proximal pausing of RNA polymerase II (Pol II) by recruiting PAF1 complex to the provirus. Following latency reversal,MLL1 complex competitively displaces PAF1 from the provirus through casein kinase II (CKII)-dependent association with LEDGF/p75. Depleting or pharmacologically inhibiting CKII prevents PAF1 dissociation and abrogates the recruitment of both MLL1 and Super Elongation Complex (SEC) to the provirus,thereby impairing transcriptional reactivation for latency reversal. These findings,therefore,provide a mechanistic understanding of how LEDGF/p75 coordinates its distinct regulatory functions at different stages of the post-integrated HIV life cycles. Targeting these mechanisms may have a therapeutic potential to eradicate HIV infection. View Publication

Background: Differences in DNA methylation have been reported in B and T lymphocyte populations,including CD4+ T cells,isolated from rheumatoid arthritis (RA) patients when compared to healthy controls. CD4+ T cells are a heterogeneous cell type with subpopulations displaying distinct DNA methylation patterns. In this study,we investigated DNA methylation using reduced representation bisulfite sequencing in two CD4+ T cell populations (CD4+ memory and na{\{i}}ve cells) in three groups: newly diagnosed disease modifying antirheumatic drugs (DMARD) na{\"{i}}ve RA patients (N = 11) methotrexate (MTX) treated RA patients (N = 18) and healthy controls (N = 9) matched for age gender and smoking status. Results: Analyses of these data revealed significantly more differentially methylated positions (DMPs) in CD4+ memory than in CD4+ na{\""{i}}ve T cells (904 vs. 19 DMPs) in RA patients compared to controls. The majority of DMPs (72{\%}) identified in newly diagnosed and DMARD na{\""{i}}ve RA patients with active disease showed increased DNA methylation (39 DMPs) whereas most DMPs (80{\%}) identified in the MTX treated RA patients in remission displayed decreased DNA methylation (694 DMPs). Interestingly we also found that about one third of the 101 known RA risk loci overlapped (±500 kb) with the DMPs. Notably introns of the UBASH3A gene harbor both the lead RA risk SNP and two DMPs in CD4+ memory T cells. Conclusion: Our results suggest that RA associated DNA methylation differences vary between the two T cell subsets but are also influenced by RA characteristics such as disease activity disease duration and/or MTX treatment.""" View Publication

Circulating tumor cells (CTC) play important roles in various cancers; however,few studies have assessed their clinical utility in neuroendocrine tumors. This study aimed to prospectively evaluate the prognostic value of CTC counts in Asian patients with neuroendocrine tumors before and during anti-cancer therapy. Patients who were diagnosed with unresectable histological neuroendocrine tumors between September 2011 and September 2017 were enrolled. CTC testing was performed before and during anti-cancer therapy using a negative selection protocol. Chromogranin A levels were also assessed. Univariate and multivariate Cox's proportional hazard model with forward LR model was performed to investigate the impact of independent factors on overall survival and progression-free survival. Kaplan-Meier method with log-rank tests were used to determine the difference among different clinicopathological signatures and CTC cutoff. The baseline CTC detection rate was 94.3{\%} (33/35). CTC counts were associated with cancer stages (I-III vs. IV,P = 0.015),liver metastasis (P = 0.026),and neuroendocrine tumor grading (P = 0.03). The median progression-free survival and overall survivals were 12.3 and 30.4 months,respectively. In multivariate Cox regression model,neuroendocrine tumors grading and baseline CTC counts were both independent prognostic factors for progression-free survival (PFS,P = 0.005 and 0.015,respectively) and overall survival (OS,P = 0.018 and 0.023,respectively). In Kaplan-Meier analysis,lower baseline chromogranin A levels were associated with longer PFS (P = 0.024). Baseline CTC counts are associated with the clinicopathologic features of neuroendocrine tumors and are an independent prognostic factor for this malignancy. View Publication

The inability of Nef to downmodulate the CD3-T cell receptor (TCR) complex distinguishes HIV-1 from other primate lentiviruses and may contribute to its high virulence. However,the role of this Nef function in virus-mediated immune activation and pathogenicity remains speculative. Here,we selectively disrupted this Nef activity in SIVmac239 and analyzed the consequences for the virological,immunological,and clinical outcome of infection in rhesus macaques. The inability to downmodulate CD3-TCR does not impair viral replication during acute infection but is associated with increased immune activation and antiviral gene expression. Subsequent early reversion in three of six animals suggests strong selective pressure for this Nef function and is associated with high viral loads and progression to simian AIDS. In the absence of reversions,however,viral replication and the clinical course of infection are attenuated. Thus,Nef-mediated downmodulation of CD3 dampens the inflammatory response to simian immunodeficiency virus (SIV) infection and seems critical for efficient viral immune evasion. View Publication

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy,but the overall metabolic properties of HSCs remain elusive. Using combined approaches,including single-cell RNA sequencing (RNA-seq),we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed,but not quiescent,HSCs relied readily on glycolysis. Notably,in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant. View Publication

Overcoming drug resistance and targeting cancer stem cells remain challenges for curative cancer treatment. To investigate the role of miRNAs in regulating drug resistance and leukemic stem cell (LSCs) fate,we performed global transcriptome profiling in treatment-na{\{i}}ve chronic myeloid leukemia (CML) stem/progenitor cells and identified that miR-185 levels anticipate their response to ABL tyrosine kinase inhibitors (TKIs). miR-185 functions as a tumor suppressor; its restored expression impaired survival of drug-resistant cells sensitized them to TKIs in vitro and markedly eliminated long-term repopulating LSCs and infiltrating blast cells conferring a survival advantage in pre-clinical xenotransplantation models. Integrative analysis with mRNA profiles uncovered PAK6 as a crucial target of miR-185 and pharmacological inhibition of PAK6 perturbed the RAS/MAPK pathway and mitochondrial activity sensitizing therapy-resistant cells to TKIs. Thus miR-185 presents as a potential predictive biomarker and dual targeting of miR-185-mediated PAK6 activity and BCR-ABL may provide a valuable strategy for overcoming drug resistance in patients." View Publication

S100A12 is a calcium-binding protein of the S100 subfamily of myeloid-related proteins that acts as an alarmin to induce a pro-inflammatory innate immune response. It has been linked to several chronic inflammatory diseases,however its role in the common oral immunopathology periodontitis is largely unknown. Previous in vitro monoculture experiments indicate that S100A12 production decreases during monocyte differentiation stages,while the regulation within tissue is poorly defined. This study evaluated S100A12 expression in monocyte subsets,during monocyte-to-macrophage differentiation and following polarization,both in monoculture and in a tissue context,utilizing a three-dimensional co-culture oral tissue model. Further,we explored the involvement of S100A12 in periodontitis by analyzing its expression in peripheral circulation and gingival tissue,as well as in saliva. We found that S100A12 expression was higher in classical than in non-classical monocytes. S100A12 expression and protein secretion declined significantly during monocyte-to-macrophage differentiation,while polarization of monocyte-derived macrophages had no effect on either. Peripheral monocytes from periodontitis patients had higher S100A12 expression than monocytes from controls,a difference particularly observed in the intermediate and non-classical monocyte subsets. Further,monocytes from periodontitis patients displayed an increased secretion of S100A12 compared with monocytes from controls. In oral tissue cultures,monocyte differentiation resulted in increased S100A12 secretion over time,which further increased after inflammatory stimuli. Likewise,S100A12 expression was higher in gingival tissue from periodontitis patients where monocyte-derived cells exhibited higher expression of S100A12 in comparison to non-periodontitis tissue. In line with our findings,patients with severe periodontitis had significantly higher levels of S100A12 in saliva compared to non-periodontitis patients,and the levels correlated to clinical periodontal parameters. Taken together,S100A12 is predominantly secreted by monocytes rather than by monocyte-derived cells. Moreover,S100A12 is increased in inflamed tissue cultures,potentially as a result of enhanced production by monocyte-derived cells. This study implicates the involvement of S100A12 in periodontitis pathogenesis,as evidenced by increased S100A12 expression in inflamed gingival tissue,which may be due to altered circulatory monocytes in periodontitis. View Publication