Inhibition of this transporter leads to reduction of cystine uptake, cyst(e)ine deprivation, subsequent depletion of the versatile antioxidant glutathione, and reactive lipid species-dependent death. Accordingly, pharmacological inhibitors of the Xc− transporter can also induce neuronal cell death raising concerns about toxicity in the CNS and PNS if these agents are used for chemotherapy. Here, we show that ferroptotic death induced by the canonical ferroptosis inducer erastin is similar in HT1080 fibrosarcoma cells and primary cortical neurons although cell death is mediated more potently in cancer cells. Reducing the toxicity of ferroptosis inducers will require, among other things, the identification of agents that protect neurons from ferroptosis but exacerbate it in tumor cells. Although we show that a number of agents known to block ferroptosis in primary mouse neurons also inhibit ferroptosis in fibrosarcoma cells, class I histone deacetylase (HDAC) inhibitors selectively protect neurons while augmenting ferroptosis in cancer cells. Our results further suggest that cell death pathways induced by erastin in these two cell types are statistically identical to each other and identical to oxidative glutamate toxicity in neurons, where death is also mediated via inhibition of Xc− cystine transport. Together, these studies identify HDACs inhibitors as a novel class of agents to augment tumor suppression by ferroptosis induction and to minimize neuronal toxicity that could manifest as peripheral neuropathy or chemo brain.Ferroptotic death is a mechanism for tumor suppression by pharmacological inhibitors that target the X A major challenge in cancer chemotherapy is to effectively kill tumor cells while preserving healthy tissue. The nervous system is particularly vulnerable to side effects by anti-cancer agents. Agents that induce a recently identified type of cell death, called ferroptosis, are widely being considered for cancer treatment. However, precise understanding of how ferroptosis induction in cancer cells may simultaneously thwart function or viability of post-mitotic neurons is essential in defining the efficacy and toxicity of these agents. We show that mechanisms of ferroptotic cell death in cancer cells are similar to those in neurons. We leverage prior studies of ferroptosis in neurons to identify histone deacetylase inhibitors as agents that enhance chemotherapy-induced ferroptosis of tumors while inhibiting ferroptosis in neurons.c−). Cyst(e)ine depletion leads to decreased levels of glutathione  or peripheral nervous system  toxicities . These tTo understand the potential toxicities of erastin in CNS neurons, we here examine the signaling pathways engaged by erastin in post-mitotic cortical neurons and compare them to those activated in HT1080 fibrosarcoma cells.1080), cyclosporine A (C-6000), Olaparib (O-9201), SB203580 (S-3400), SP600125 (S-7979), and U0126 (U-6770) were obtained from LC Laboratories. Laminin (CC095), GSK’872 (530389), Necrostatin-1 inactive (480066), Necrosulfonamide (480073) mouse tumor necrosis factor-α (GF027), U0124 (662006), and rabbit anti-acetylated Histone H4  antibody were from Millipore; Boc-DON-Gln-Ile-Val-OMe (B003), 1,3-dimethyl-4,5-diphenyl-2-[(2-oxopropyl)thio]imidazolium trifluorosulfonic acid salt (D004) from Zedira GmbH; 3--2,5-diphenyltetrazolium bromide  from Promega; erastin (S7242) and entinostat  from Selleck Chemicals. Actinomycin D (A1410), Chloroquine (C6628), cycloheximide (01810), Cystamine dihydrochloride (C8707), Deferoxamine (D9533), Ferrostatin-1 (SML0583), Homocysteate (H9633), Mithramycin A (M6891), N-acetylcysteine (A7250), rapamycin (R8781), sodium butyrate (303410), protease inhibitor cocktail (P8340), poly-d-lysine (P6407), poly-l-lysine (P4704), N1 supplement (N6530), collagenase (C9697), EGTA (E0396), sodium orthovanadate (S6508), mouse anti-β-actin , and Tween 20 (P7949) were obtained from Sigma-Aldrich. EDTA (E177) was obtained from Amresco. Triton X-100 (161-0407), DC Protein Assay Kit I (5000111), Quick Start Bradford Reagent (500-0205), and Protein Dual Color Standard (161-0374) were purchased from Bio-Rad. DMEM (11965118), DMEM/nutrient mixture F-12 (10565-018), MEM GlutaMAX Supplement (41090101), MEM non-essential amino acids (11140050), fetal bovine serum (16140071), horse serum (26050088), penicillin-streptomycin (15140163), live/dead assay (L3224), NuPAGE 4% and 12% Bis-Tris protein gels (NP0335 and NP0336), MES SDS Running Buffer (NP0002), TaqMan c-Myc (Hs00153408), p21 (Hs00355782), HDAC1 (Hs00606262_g1), HDAC2 (Hs00231032_m1), HDAC3 (Hs00187320_m1), HDAC8 (Hs00954353_g1), GAPDH (4332649) human primers, HDAC1 (Mm02391771), HDAC2 (Mm00515108), HDAC3 (Mm00515916), HDAC8 (Mm01224980_m1) mouse primers, mouse β-actin endogenous control VIC (4352341E), TaqMan RNA-to-CT 1-Step Kit (4392656), and MicroAmp 96-well Reaction Plates (4346906) were purchased from ThermoFisher Scientific. Laemmli SDS Sample Buffer (BP-110R), Transfer Buffer (BP-190), and Tris-Buffered Saline (BM-300) were obtained from Boston BioProducts. Methanol (BDH1135) was purchased from VWR. Mouse anti-Histone H4  antibody was from Cell Signaling Technology. Rabbit anti-phospho-S166 RIP1 antibody (1:2000) was provided by P.J.G., J.B., and J.F. (GlaxoSmithKline). Nitrocellulose membrane 0.2 µm (10600001) was from GE Healthcare. Odyssey Blocking Buffer (927-40010), goat anti-rabbit 680RD , and goat anti-mouse 800CW  were purchased from LI-COR Biosciences. NucleoSpin RNA isolation kit (740955) was obtained from Clontech. Eagle’s Minimum Essential Medium (30-2003) was from American type Culture Collection.Apicidin , DPQ (14450), and Nullscript (16433) were obtained from Cayman Chemical. 3-Methyladenine (BML-AP502-0025), Mdivi-1 (BML-CM127-0010), Necrostatin-1 (BML-AP309-0020), Scriptaid (BML-GR326-0005), Trolox (ALX-270-267-M100), and z-VAD-fmk (ALX-260-138-R100) were purchased from Enzo Life Sciences. Bafilomycin A1  and water ad libitum.All animal procedures were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee  and conducted in accordance with the NIH d-lysine-coated plates in minimum essential medium containing 10% fetal bovine serum, 5% horse serum, and 1% penicillin/streptomycin .Primary cortical neurons were obtained from CD-1/ICR mice of either sex at embryonic day 14.5. Briefly, cortices were dissected, homogenized, and plated in poly-l-lysine/laminin-coated plates in DMEM/nutrient mixture F-12 supplemented with 1× N1 and 10% horse serum .Primary dorsal root ganglia (DRG) neurons were obtained from C57BL/6 mice of either sex at 6 weeks of age. Briefly, the spinal column was isolated by dissection and the spinal cord removed by hydraulic extrusion. The spinal cord was split longitudinally and the DRGs located and removed. DRGs were dissociated with collagenase, plated onto poly-Immortalized hippocampal neuroblasts (HT22 cells) were cultured in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin . HT1080 cells were obtained from American type Culture Collection and cultured in DMEM containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% non-essential amino acids . Hep3B cells were obtained from American type Culture Collection and cultured in Eagle’s minimum essential medium containing 10% fetal bovine serum and 1% penicillin/streptomycin . SH-SY5Y cells were also obtained from American type Culture Collection and cultured in DMEM/nutrient mixture F-12 containing 10% fetal bovine serum and 1% penicillin/streptomycin . Cell lines were treated at 24 h when density reached 70% confluency.2 atmosphere.All cells were cultured at 37°C in a humidified 5% COCell viability was determined at 22–26 h following erastin or glutamate analog homocysteate (HCA) exposure using MTT assay, a colorimetric assay of cell metabolic activity. We measured the plates at SpectraMax Plus Microplate Reader using SoftMax Pro v4.7.1 (both Molecular Devices). The results of population, quantitative assays of cell viability (MTT) were verified by qualitative LIVE/DEAD assay and fluorescence microscopy at Nikon Eclipse TS100 microscope using Nikon DS-L3 (Nikon Instruments).m :25 Tris, pH 7.4, 100 NaCl, 1 EGTA, 1% Triton X-100, protease inhibitors, 2.5 sodium orthovanadate) except in case of the quantification of histones, where RIPA-B lysis buffer  was used. We electrophoresed the samples under reducing conditions on NuPAGE gels and transferred them to a nitrocellulose membrane. Antibodies against phospho-S166 RIP1, acetylated histone H4, total histone H4, and β-actin were incubated overnight at 4°C. Secondary antibodies were incubated for 1 h at room temperature. We detected the proteins using Odyssey infrared imaging system (LI-COR Biosciences).Protein extracts were prepared using 1% Triton buffer , p21 (Hs00355782), HDAC1 (Hs00606262_g1), HDAC2 (Hs00231032_m1), HDAC3 (Hs00187320_m1), HDAC8 (Hs00954353_g1), and mouse HDAC1 (Mm02391771), HDAC2 (Mm00515108), HDAC3 (Mm00515916), and HDAC8 (Mm01224980_m1) at a 7500 Real-Time PCR System (Applied Biosystems). Expression levels were normalized to mouse β-actin endogenous control.post hoc Bonferroni test. In case one of the criteria was not met, the Kruskal–Wallis test was performed followed by the post hoc Mann–Whitney U test with α correction according to Bonferroni to adjust for the inflation of type I error due to multiple testing. Data are represented as mean ± SD except for nonparametric data, where medians are given. A value of p < 0.05 was considered statistically significant. For the Kruskal–Wallis test followed by Mann–Whitney U, p = 0.05/k was used, with k as the number of single hypotheses. K = 2 for gene expression experiments (comparison of 2 different concentrations vs vehicle-treated cells), k = 4 (comparison of 3 different concentrations vs vehicle-treated cells) for all nonparametric data of drug treatments, except for Necrostatin-1, Scriptaid, and U0126, where k = 12 , and pRIP1, where k = 9 . Thus α = 0.025 for two comparisons, α = 0.0125 for four comparisons, α = 0.0056 for 9 comparisons, and α = 0.0042 for 12 comparisons was considered statistically significant. To analyze contingency tables, Fisher’s exact test was used. Detailed statistical analyses can be found in the extended data (SCR_002865).All data represent biological replicates. For the MTT assay, each biological replicate is the mean of four technical replicates. Normality was evaluated with the Kolmogorov–Smirnov test and variance homogeneity using the Levené test. When data were normally distributed and variance was homogeneous, we performed one-way ANOVA followed by the ded data . All sta10.1523/ENEURO.0263-18.2019.f3-1Figure 3-1Figure 3-1, DOCX file.Statistical data on ferroptosis inhibitors in HT1080 cells and primary cortical neurons. Download 10.1523/ENEURO.0263-18.2019.f5-1Figure 5-1Figure 5-1, DOCX file.Statistical data on apoptosis inhibitors in HT1080 cells and primary cortical neurons. Download 10.1523/ENEURO.0263-18.2019.f7-1Figure 7-1Figure 7-1, DOCX file.Statistical data on parthanatos and necroptosis inhibitors in HT1080 cells and primary cortical neurons. Download 10.1523/ENEURO.0263-18.2019.f9-1Figure 9-1Figure 9-1, DOCX file.Statistical data on autophagy inhibitors in HT1080 cells and primary cortical neurons. Download 10.1523/ENEURO.0263-18.2019.f10-1Figure 10-1Figure 10-1, DOCX file.Statistical data on levels of pRIP1 in erastin- and glutamate analog (HCA)-induced cell death. Download 10.1523/ENEURO.0263-18.2019.f13-1Figure 13-1Figure 13-1, DOCX file.Statistical data on cell death inhibitors in erastin-induced cell death in HT1080 cells. Download 10.1523/ENEURO.0263-18.2019.f13-2Figure 13-2Figure 13-2, DOCX file.Statistical data on gene expression after mithramycin treatment in HT1080 cells. Download 10.1523/ENEURO.0263-18.2019.f13-3Figure 13-3Figure 13-3, DOCX file.Statistical data on Scriptaid and Nullscript in erastin-induced death in primary cortical neurons. Download 10.1523/ENEURO.0263-18.2019.f13-4Figure 13-4Figure 13-4, DOCX file.Statistical data on HDAC gene expression in primary cortical neurons versus HT1080 cells. Download 10.1523/ENEURO.0263-18.2019.f14-1Figure 14-1Figure 14-1, DOCX file.Statistical data on Scriptaid in erastin-induced cell death in SH-SY5Y and Hep3B cells. Download N-acetylcysteine, Trolox), iron , and ERK signaling ine deprivation  and is om) to study the mechanisms of death led to a 95% reduction in cell survival  via a non-receptor-mediated mechanism involving inhibition of the System Xc− (cystine/glutamate) antiporter ine or glutathione depletion has been elucidated as an tiporter A. This mtiporter , b. To dThere are many other modes of cell death . ApoptosFigs. 4–Unexpectedly, several modulators of autophagy , with no activity toward RIP1, that did not block erastin- or glutamate analog (HCA)-induced toxicity. Moreover, as RIP1 kinase activity is required to execute necroptosis , we assep = 0.202), despite the ability of the inducers of autophagy or the inhibitors of necroptosis to prevent “ferroptosis” at the lower dose (1 µM) in HT1080 cells. Second, erastin toxicity in HT1080 fibrosarcoma cells and in neurons can be considered mechanistically similar . Similarly, erastin and glutamate analog (HCA) toxicity in neurons can be considered mechanistically similar .To elucidate whether erastin- and glutamate analog (HCA)-induced toxicities are similar or distinct modes of cell death A, we perp = 0.751). Convergence of these distinct modes of cell death in a single cell death paradigm has not been described so far.Collectively, these findings suggest that erastin induces cell death in both cancer cells and neurons through ferroptotic, autophagic, and necroptotic pathways and that they are not different from what is known from the established glutathione depletion model referred to incorrectly as apoptosis  or correBecause erastin toxicity in HT1080 cells and glutamate analog (HCA)-induced glutathione depletion in neurons induce ferroptotic cell death, we hypothesized that prior knowledge about glutamate analog (HCA)-induced ferroptosis in neurons should inform the points of convergence and divergence with erastin-induced ferroptosis in HT1080 fibrosarcoma cells. We therefore investigated whether erastin-induced ferroptosis in HT1080 can be abolished or exacerbated by the chemical inhibitors known to be effective in glutamate analog (HCA)-induced neuronal ferroptosis , 13A.de novo transcription via the leucine zipper transcription factor ATF4 leading to upregulation of putative pro-death genes such as Trib3, Chop, and Chac1 -induced ferroptosis in neurons requires nd Chac1 . Recentlnd Chac1 . As expend Chac1 , 13A.in vitro and extend survival in vivo in an HD model, where ferroptosis has also been implicated (m) abrogated erastin-induced death in cancer cells -induced ferroptosis plicated , b. Mither cells , 13A. Weer cells .Transglutaminases are established transcriptional targets for Huntington’s disease  and haveAnother class of agents with known antitumor activity shown to abrogate HCA-induced ferroptosis are class I HDAC inhibitors , 2014. SWe then hypothesized that the expression of distinct HDAC isoforms in neurons versus cancer cells explains this difference. We therefore assessed the gene expression of HDACs from class I and found that HT1080 cells and primary neurons express HDAC1, 2, and 3. Surprisingly, only HT1080 cells express HDAC8, raising the intriguing possibility that HDAC 8 expression may explain the differential effects of HDAC inhibitors between HT1080 cells and primary neurons E.50 of erastin for each cell line). We found that 2.5-10 µm Scriptaid exacerbated erastin-induced cell death in SH-SY5Y cells, whereas the negative control did not. However, it also decreased viability of SH-SY5Y cells without erastin treatment  induces death in cultured CNS neurons compared with erastin-induced death in cancer cells. The Xc− transporting agency is a chloride-dependent transporter that exchanges cystine for glutamate. Cystine is converted intracellularly to cysteine, which is used for protein synthesis and synthesis of the versatile antioxidant glutathione. Depletion of cyst(e)ine leads to depletion of glutathione and reactive lipid species-induced death now defined as ferroptosis.The objective of this study was to understand the mechanisms by which erastin -induced ferroptosis/oxytosis in neurons can be applied to ferroptosis in cancer cells , 13. Indc− transport inhibitor like erastin, has been shown to not only inhibit glial-derived tumor (glioma) growth but also to inhibit neuronal excitotoxic death and seizures associated with these tumors  where there is clear sensitivity of the glioma tumor to glutathione depletion but no effect on neurons or astrocytes. Indeed, studies here clearly demonstrate that cortical neurons are sensitive to Xc− transport inhibition with an LD50 5-fold higher than those required to kill 50% of the HT1080 cells. Second, they did not evaluate toxicity to peripheral neurons or central neurons directly, still leaving the question open whether Xc− inhibition systemically can lead to chemotherapy-induced neuropathy or chemo brain. Accordingly, apparent toxicity may occur under circumstances where drug concentrations used to kill tumor cells are higher and begin to approach the susceptibility of neurons. HDAC inhibitors in combination with erastin appear to be a viable strategy to minimize toxicity as they would reduce the concentration of erastin or sulfasalazine required for killing but would provide for neuroprotection from ferroptosis in their own right.However, several observations potentially reconcile our results with seminal studies from Sontheimer et al. First, they evaluated the effects of XIn conclusion, cell death pathways in cancer cells and neurons exposed to glutathione depletion are similar as judged by the Fisher’s tests. Our data suggest that HDAC inhibitors are promising therapeutic agents for combinatorial cancer chemotherapy to enhance chemotherapy-induced ferroptosis of tumors while inhibiting ferroptosis in neurons. The current study further identifies agents, some of which are known antitumor drugs, which would prevent erastin-induced toxicity in neurons  and in cancer cells suggesting that these are not good combinatorial therapies to optimally kill tumor cells while preserving CNS and PNS neurons.


Caenorhabditis elegans to study the role of deuterated polyunsaturated fatty acids (D-PUFAs). Deuterated trilinolenin [D-TG(54:9)] was sufficient to prevent the accumulation of lipid peroxides and to reduce the accumulation or ROS. Moreover, D-TG(54:9) significantly extended the lifespan of worms under normal and oxidative stress conditions. These findings demonstrate that D-PUFAs can be used as a food supplement to decelerate the aging process, resulting in extended lifespan.Chemically reinforced essential fatty acids (FAs) promise to fight numerous age-related diseases including Alzheimer’s, Friedreich’s ataxia and other neurological conditions. The reinforcement is achieved by substituting the atoms of hydrogen at the bis-allylic methylene of these essential FAs with the isotope deuterium. This substitution leads to a significantly slower oxidation due to the kinetic isotope effect, inhibiting membrane damage. The approach has the advantage of preventing the harmful accumulation of reactive oxygen species (ROS) by inhibiting the propagation of lipid peroxidation while antioxidants potentially neutralize beneficial oxidative species. Here, we developed a model system to mimic the human dietary requirement of omega-3 in  Sensitive to oxidative damage, the brain consumes around 20% of oxygen despite making only 2% of body weight. The brain is rich in polyunsaturated fatty acids (PUFAs) and oxygen in the lipid bilayer is particularly high reaching millimolar levels . This tiSenescence is a major cause of age-related diseases, correlating with an accumulation of ROS . Enhancein vivo, and supplementation of dietary antioxidants might compromise this critical equilibrium  methylene group, which is followed quickly by a series of transformations, generating toxic end-products . During ilibrium . This mailibrium .Saccharomyces cerevisiae, deuterated PUFAs (D-PUFAs) have been shown to reduce oxidative stress  and α-linolenic acid (ALA) FAs . Thus, ae stress  by protee stress . In addie stress  and in Fe stress . More reAldh2-/- . This reAldh2-/- . FriedreAldh2-/- . More thAldh2-/- . HoweverC. elegans. This organism was recently reported to be sensitive to deuterated lysine  which strongly affected its development  mutant worm which is also sensitive to the oxidative stress agent paraquat. Triglyceride with three omega-3 PUFAs, trilinolenin [TG(54:9)], was sufficient to induce lipid peroxidation while deuterated TG(54:9) [D-TG(54:9)] was protective. D-TG(54:9) reduced the oxidative stress response in addition of significantly extending the lifespan of fat-1(lof) under normal and oxidative stress conditions. These findings demonstrate that D-TG(54:9) is adequate to prevent the propagation of ROS-induced molecular damages resulting in a significant extension of lifespan.In this study, we characterized the role of D-PUFAs against ROS in the multicellular model organism elopment . To mimiP-values were calculated using one-way ANOVA with Tukey’s test or log-rank test for lifespan, unless otherwise indicated and reported as P-values. All statistical tests were performed using GraphPad Prism 7 software.Error bars indicate standard error of the mean (SEM), calculated from at least three biological replicates, unless otherwise indicated. C. elegans methods as previously described , mev-1(tk22), gst-4p::GFP::NLS(cl2166), sod-3p::GFP(cf1553) and bacteria strains OP50 were gifted from the Caenorhaditis Genetics Center. Trilinolenin [TG(54:9)] was obtained from Nu-Chek Prep and deuterated at bis-allylic position as previously described , 0.48 mM D-TG(54:9) or carrier. Approximately 10,000 L4 to young adult worms were harvested and washed thoroughly with M9 buffer and lyophilised overnight . FAs were esterified to fatty acid methyl esters (FAME) with 300 μl of 1.25 M HCl-methanol for 1 h at 80°C. FAMEs were extracted three times with 1 ml of hexane. Combined extracts were dried under nitrogen, resuspended in 100 μl hexane. FAMEs were separated by gas chromatography with flame ionization detector  using an ULBON HR-SS-10 50 m × 0.25 mm column . Supelco 37 component FAME mix was used to identify corresponding FAs . Data was normalized using internal standard pentadecanoic acid (C15:0) and worm dry weight.Synchronized L1 mev-1 mutants were transferred to NGM plates containing 0.1 mM BHT, 0.48 mM TG(54:9), 0.48 mM D-TG(54:9) or carrier for 54 h, after which they were transferred on supplemented plates with 8 mM paraquat when indicated for 24 h. Approximately 1,000 L4 to adult worms were harvested and washed thoroughly with M9 buffer, resuspended in 300 μl RIPA buffer , lysed with 1 mm silica beads by bead beating. Protein concentration was carried out using the Bicinchoninic Acid (BCA) Protein Assays kit following manufacturer’s protocol . Assays for lipid peroxidation, using the thiobarbituric acid reactive substrate (TBARS) kit were performed following manufacturer’s protocol .581-591-C11) solution and stained for 30 min and washed three times with M9. Images were captured using confocal fluorescence microscope Zeiss LSM 710 microscope with a 20 × objective (Carl Zeiss MicroImaging). Oxidized and non-oxidized BODIPY581-591-C11 were excited at 488 and 568 and images were collected from emission at 530(30) and 590(30) nm, respectively. Fluorescence signal ratio of oxidized to non-oxidized BODIPY was normalized to carrier.Synchronized L1 mev-1 animals were transferred to NGM plates containing 0.1 mM BHT, 0.48 mM TG(54:9), 0.48 mM D-TG(54:9) or carrier and supplemented with 5 mM paraquat when indicated. L4 to young adult worms were transferred to 10 μM BODIPY 581/591 undecanoic acid (BODIPYgst-4p::GFP or sod-3p::GFP worms were transferred to NGM plates containing 0.48 mM TG(54:9), 0.48 mM D-TG(54:9) or carrier and supplemented with 5 mM paraquat when indicated. To quantify GFP signal, worms were immobilized with 25 mM tetramisole and mounted on 2% agarose pad. Images were captured using Zeiss Axiovert 200 M fluorescence microscope with a 20 × objective. Images were stitched, and total fluorescence were quantified using Fiji ImageJ software.Synchronized L1 fat-1(lof) animals were transferred to NGM plates containing 0.48 mM TG(54:9), or 0.48 mM D-TG(54:9) and supplemented with 5 μM 5-fluoro-2’-deoxyuridine  to facilitate the counting of the eggs. The fertility of the worms was scored by counting eggs on day 5 and 6 after the transfer of the adults to a new plate.Synchronized L1 WT and C. elegans synthesize both LA and ALA consequently we selected omega-3 fatty acid desaturase fat-1 mutant animal to mimic the human dietary requirement of ALA (2∙-). In turn, hydroperoxyl radicals HO2∙ modify PUFAs through oxidative damage in a chain-propagation fashion (C. elegans (fat-1(lof) worms. As expected, high concentration of 1 mM paraquat significantly reduced the lifespan of fat-1(lof) worms compared to carrier (fat-1(lof) worms are sensitive to the production of ROS by the toxic level of paraquat. We also subjected fat-1(lof) worms to the antioxidant BHT but the lifespan was unchanged compared to carrier. This result suggests that fat-1(lof) basal level of ROS is not harmful and neutralising ROS further with antioxidant does not influence fat-1(lof) longevity.In human, FAs lineloic acid  and α-linolenic acid  are essentials . In contt of ALA . We test fashion . As low  elegans  while hi elegans , we carr carrier . This re-/-Aldh2 (2 group between the two double bonds was effective at 97.5% (data not shown) using our recently reported site-specific deuteration of polyunsaturated alkenes method (C. elegans ingests and metabolizes D-TG(54:9) as well as non-deuterated TG(54:9), we fed L1 larvae fat-1(lof) mutant with standard diet on NGM plates supplemented with 0.48 mM TG(54:9), D-TG(54:9), or the carrier (0.1% Triton X-100 in PBS). C. elegans is typically fed E. coli OP50 bacteria in which PUFAs are naturally absent but can be easily incorporated if supplemented to the media (fat-1(lof) fed D-TG(54:9) while the other changes were statistically non-significant compared to carrier  mutant by measuring its fertility which is diminished by ROS  mutant with standard diet on NGM plates supplemented with 0.48 mM TG(54:9), D-TG(54:9), or the carrier. On carrier, fat-1(lof) mutant laid a significant lower amount of eggs compared to WT (fat-1(lof) mutant, fed TG(54:9), laid a similar amount of eggs per worm compared to fat-1(lof) mutant on carrier. To our surprise, fat-1(lof) mutant, fed D-TG(54:9), laid a similar amount of eggs per worm compared to WT. This result suggests that D-TG(54:9) promotes fertility in fat-1(lof) mutant while TG(54:9) might promote lipid peroxidation.Next, we asked if D-TG(54:9) is toxic to d by ROS . As Δ9 fertility , we monied to WT . Similarmev-1(lof) mutant worms (mev-1(lof) mutants were grown on NGM plates seeded with bacteria supplemented with carrier, BHT, TG(54:9), or D-TG(54:9). Subsequently, L4/young adult mev-1(lof) mutants were exposed to 24 h of paraquat. Paraquat alone was insufficient to significantly increase the accumulation of lipid peroxide compared to carrier alone (mev-1(lof) mutants grown on TG(54:9) and subjected to paraquat exhibited a dramatic increase in lipid peroxides compared to paraquat alone and to TG(54:9) in the absence of paraquat. This result reinforces the role of PUFAs in catalyzing the propagation of ROS through lipid peroxidation . The sensor emission peak shifts from 590 to 510 nm when the polyunsaturated butadienyl domain is oxidized (mev-1(lof) mutants were grown and treated as for the TBARS assay. In contrast to the TBARS assay, paraquat alone was sufficient to significantly induce lipid peroxidation (581/591-C11 is more sensitive than the TBARS assay to monitor lipid peroxidation in C. elegans as previously reported in different cell types (mev-1(lof) mutants. D-TG(54:9) was sufficient to protect mev-1(lof) mutants against paraquat-induced lipid peroxidation. As the BODIPY-C11 lipid peroxidation reporter assay yielded small differences, BODIPY possibly failed to be incorporated evenly through the animal and perhaps not sufficiently where most of lipid peroxidation occurs. Together with the TBARS assay, these results demonstrate that D-PUFA is sufficient to prevent lipid peroxidation in C. elegans.To further assess the role of D-TG(54:9) in preventing the formation of lipid peroxides, we monitored oxidized . mev-1(lxidation . This sull types . In contin vivo. We monitored the expression of the reporter protein GFP under the promotor sod-3 (sod-3p::GFP) (sod-3p::GFP was observed across the different conditions (gst-4 (gst-4p::GFP::NLS) (gst-4p::GFP::NLS worms were grown with triacylglycerol and paraquat as per the TBARS assay. The expression of gst-4p::GFP::NLS was significantly increased in the presence of paraquat and further increase with the addition of TG(54:9) (To further assess the role of D-TG(54:9) in preventing the propagation of lipid peroxide, we monitored the oxidative stress response 3p::GFP) . SOD-3 i3p::GFP) . These wnditions . As thisFP::NLS) . GST-4 iFP::NLS) . GlutathFP::NLS) . gst-4p:TG(54:9) . On the fat-1(lof) worms fed normal diet supplemented with either TG(54:9) or D-TG(54:9). D-TG(54:9) significantly extended the lifespan of worms compared to the supplementation with TG(54:9) (fat-1(lof) worms, we exposed the animals to paraquat from L1 stage. As expected, the lifespan of fat-1(lof) worms exposed to paraquat was not significantly extended by TG(54:9) (fat-1(lof) worms are unable to synthesize several PUFAs (fat-1(lof) worms exposed to paraquat was further extended with D-TG(54:9) compared to paraquat alone and to TG(54:9) in combination with paraquat. This result indicates that D-TG(54:9) is sufficient to promote longevity. Potentially, supplementing a human diet with D-PUFAs might be sufficient to decelerate aging and to prevent the progression of age-associated diseases.As D-TG(54:9) reduces oxidative stress, we carried out a lifespan assay of TG(54:9) . This suTG(54:9) . As fat-al PUFAs , TG(54:9Chemically reinforced essential FAs have the potential to target age-related diseases such as Alzheimer’s, Friedreich’s ataxia and other neurological conditions. Substituting the atoms of hydrogen at the bis-allylic methylene of essential polyunsaturated FAs with isotope deuterium prevents substantial chemical damage. Recently, we developed a novel synthesis approach to reinforced natural FAs , which cin vivo, and supplementation of dietary antioxidants might compromise this delicate equilibrium. More importantly, as PUFAs belong to the group of essential nutrients that must be supplied with diet, it was proposed that PUFAs with deuterated bis-allylic methylenes could represent a novel approach to fight against ROS-initiated attacks, leading to lessening the influence of aging and age-associated diseases on human life.During the late nineties, a large boom of research on dietary antioxidants generated hopes as potential agents to slow aging and to prevent the development of ROS-related diseases. However, mixed results emerged from studies conducted in humans . It becaC. elegans, we have demonstrated that H-PUFA promotes, while D-PUFA reduces, both lipid peroxidation and oxidative stress during paraquat-induced oxidative stress. For the first time, we have demonstrated the beneficial effect of D-PUFAs in extending longevity. In future, C. elegans could be used to validate the beneficial role of D-PUFAs in modulating the lifespan of different disease models.The protective effect of D-PUFAs against ROS has been demonstrated in yeast and in primary co-cultures of neurons and astrocytes . As unicAll datasets generated for this study are included in the manuscript and/or the supplementary files.GT conceived the study, wrote the original draft of the manuscript, and acquired the funding. CB-C and GT performed the methodology. CB-C and LW contributed to formal analysis. CB-C investigated the study. CB-C, AS, MS, and DV performed the resources. MS and GT wrote, reviewed, and edited the manuscript.MS owns stocks in Retrotope. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


MHCII-restricted antigen presentation by lymph node stromal cells is essential for regulatory T-cell proliferation and functions, and for the regulation of autoimmunity. + and CD8+ T-cell frequencies, resulting in enhanced peripheral organ T-cell infiltration and autoantibody production. The proliferation of LN-Tregs interacting with LECs increases following MHCII up-regulation by LECs upon aging or after exposure to IFN-γ, this effect being abolished in mice in which LECs lack MHCII. Overall, our work underpins the importance of LNSCs, particularly LECs, in supporting Tregs and T-cell tolerance.How lymph node stromal cells (LNSCs) shape peripheral T-cell responses remains unclear. We have previously demonstrated that murine LNSCs, lymphatic endothelial cells (LECs), blood endothelial cells (BECs), and fibroblastic reticular cells (FRCs) use the IFN-γ–inducible promoter IV (pIV) of the MHC class II (MHCII) transactivator CIITA to express MHCII. Here, we show that aging mice (>1 yr old) in which MHCII is abrogated in LNSCs by the selective deletion of pIV exhibit a significant T-cell dysregulation in LNs, including defective Treg and increased effector CD4 T-cell precursors undergo thymic negative selection, which ensures the elimination of developing T cells expressing TCR-recognizing self-Ags with excessive affinity. However, some autoreactive T cells escape this process of clonal deletion and exit the thymus to populate secondary lymphoid organs (SLOs). Therefore, additional mechanisms of T-cell tolerance are required in the periphery to avoid the development of autoimmunity. Among them, resting DCs, which constantly sample self-Ags in peripheral tissues and reach the draining LNs through the afferent lymph, present self-Ag–derived peptides to naive T cells. In the absence of danger, this phenomenon leads to clonal deletion, or anergy of autoreactive T cells . Alterna+ T-cell deletion (+ T-cell dysfunction by presenting peptide-MHC class II (MHCII) complexes acquired from DCs, LECs, BECs, and FRCs endogenously express MHCII molecules , in which MHCII molecules are loaded with peptides derived either from phagocytosis and processing of extracellular Ags  have recently been promoted to the rank of new modulators of T-cell responses. After being considered for years as simple scaffolding, forming routes, and proper environment for Ag-lymphocyte encountering, we recently learned that they also impact both DC and T-cell functions. Lymphatic endothelial cells (LECs) promote DC entry into and T-cell egress from LNs , whereasdeletion . We haveolecules . Centralular Ags , or fromular Ags . Whether−/− mice transferred with T cell isolated from LN of aging MHCII-deficient LNSC mice displayed similar immunological and clinical perturbations compared with recipient injected with age-matched control T cells, suggesting a direct link between MHCII expressed by LNSCs and the appearance of T cell–mediated signs of autoimmunity. Accordingly, upon aging or IFN-γ treatment, LECs up-regulate MHCII molecules, and interact with Treg to promote their proliferation. This phenotype is abolished in mice deficient for MHCII expression in LECs. Altogether, we prove that MHCII expression by LNSCs have a manifest impact in peripheral tolerance. Notably, LECs support self-Ag–specific T cell peripheral tolerance by promoting Treg proliferation through MHCII-restricted Ag presentation.Here, we have used genetically modified mice in which MHCII expression by non-hematopoietic cells is abrogated. Upon aging, and compared with their control counterparts, these mice exhibit an enhancement of spontaneous autoimmune processes, with enhanced T-cell activation in SLOs and effector T-cell infiltration in peripheral tissues, as well as the production of autoantibodies. In contrast, the Treg compartment is significantly impaired in SLOs. Furthermore, Rag2+ T-cell biology by presenting peptide-MHCII complexes acquired from DCs (KO) (KO mice compared with control mice (b molecules at both mRNA (KO mice (Our previous work has demonstrated that LNSCs impact CD4from DCs . AlthougDCs (KO) . As experol mice . Indeed,rol mice . DCs userol mice . Howeveroth mRNA  and prototh mRNA  levels a promoter, which is active in cTECs (Ciita pIV−/− (referred as K14tgpIVKO mice) mice compared with pIVKO mice, and to a similar extent compared with cTECs isolated from WT or control  mice  were indistinguishable from WT with respect to cell numbers and TCR V-β repertoire  depends on pIV. Because of the absence of MHCII expression by cTECs, pIV T cells . Therefoin cTECs , but notin cTECs . MHCII etg) mice . As a cotg) mice , 2016, K in SLOs . We did  in SLOs . HoweverVKO mice , for theVKO mice , and secVKO mice . Therefopertoire .K14tgpIVKO mice compared with control mice  and salivary glands (SGs) were markedly increased in K14tgpIVKO compared with K14tg control elderly (18 mo-old) mice . Therefore, peripheral tissue T-cell infiltration was increased in a variety of non-lymphoid organs in elderly K14tgpIVKO mice. In agreement with a possible autoimmune syndrome, serum obtained from 18-mo-old K14tgpIVKO mice contained a broader spectrum of autoantibodies with enhanced reactivity to proteins from several tissues compared with K14tg control mice  mice . No diffld) mice . In addicontrols . Other prol mice , indicatK14tgpIVKO mice result from enhanced T-cell activation in SLOs. Analysis of CD4+ and CD8+ T cells in LN did not show any differences between 4-mo-old K14tgpIVKO and K14tg mice (hiCD44lo) and increased frequencies of activated (CD62LloCD44hi) CD4+ and CD8+ T cells in LNs of K14tgpIVKO compared with K14tg mice  in LNs of aging K14tgpIVKO compared with K14tg control mice . In contrast, compared with control mice, LN Treg isolated from aging K14tgpIVKO mice exhibited an impaired ability to suppress the proliferation of naive CD4+ T cells in vitro at the Treg:Tnaive ratio of 1:5 . As observed in donor LNs, the frequency of Foxp3+ Tregs was significantly reduced in LNs of recipient Rag2−/− mice injected with 18-mo-old K14tgpIVKO compared with control K14tg T cells  compared with young (4 mo) mice . A slighK14tgpIVKO mice lacking MHCII expression in LNSCs. Therefore, we tested whether Treg proliferation may be affected by the loss of MHCII expression in LECs. Immunofluorescence staining on LN sections from K14tgpIVKO and control elderly showed that the frequency of proliferating  Tregs  in close interaction with LECs  was significantly increased in aging controls compared with K14tgpIVKO mice (Treg frequencies were found impaired in aging (18 mo), but not in young adult (4 mo) VKO mice .+) Tregs  in close interaction with LECs  was significantly increased in mice treated with IFN-γ compared with untreated mice  Tregs  (red arrows) in close proximity of LECs   CD4+ T cells upon IFN-γ stimulation, nor difference between K14tg controls and K14tgpIVKO mice (not shown), suggesting that the Treg compartment is specifically affected. Finally, to examine a potential direct contribution of LECs as MHCII-restricted APCs in inducing Treg proliferation, we repeated the above experiments in mice in which MHCII expression was selectively abrogated in LECs. For that, we used Prox-1-CreERT2 mice, expressing the Tamoxifen-inducible Cre recombinase under the promoter Prox-1, which is selectively expressed in adult LECs . Immunofluorescence analyses of LN sections demonstrated that the frequency of proliferating Tregs in contact with LECs 6 d after IFN-γ injection was significantly reduced in mice in which LECs do not express MHCII molecules (Prox-1-CreERT2 MHCIIfl) compared with control mice (MHCIIfl)  . In contof MHCII . In contult LECs . Prox-1-MHCIIfl) . Altoget+ T-cell dysfunction , suggesting that there is no restriction of T-cell clonality in mice lacking MHCII in LNSCs. Importantly, in this model there is no pre-existing early CD4+ T-cell bias, nor Treg function impairment. K14tgpIVKO young adult mice do not present any abnormality, excluding the possibility of a defect from thymic selection. In addition, T cells from 6- to 10-wk-old K14tgpIVKO mice were recently extensively characterized, and normal distribution of CD4+ naive, effector, and Foxp3+ T-cell subsets were observed in the thymus, spleen, and peripheral LNs , hepatocytes and liver sinusoidal endothelial cells, pancreatic β cells, or astrocytes were postulated to be capable of MHCII-mediated Ag presentation, although very little in vivo data are available on the potential impact on peripheral CD4+ T-cell responses  levels of circulating steady-state IFN-γ promote endogenous MHCII expression by LNSCs, and confer them the ability to function as competent MHCII-mediated APCs. Increased inflammatory status over time might be due to the accumulation of punctual and mild infectious processes or altered gut microbiotic composition upon aging  Prox-1-CreERT2 MHCIIfl mice have been previously described (ERT2 MHCIIfl/fl (and MHCIIfl/fl control) mice were injected with tamoxifen (i.p) twice a day for four consecutive days (2 mg/mice/d). 2 wk after the last injection, mice were treated with IFN-γ and FTY720 as described below. These mouse strains are on a C57BL/6 background and were housed and maintained under SPF conditions at the Geneva Medical School animal facility and under EOPS conditions at Charles River, France. All animal husbandry and experiments were approved by and performed in accordance with guidelines from the Animal Research Committee of the University of Geneva. Genotyping was carried out by using PCR on ear biopsies with the Phire Tissue Direct PCR Master Mix . When indicated, IFN-γ (Peprotech) was injected subcutaneously , and FTY720 (Sigma-Aldrich) (20 μg) was injected every day for 6 d before the experiment. Mice from each group were randomized before treatments.C57BL/6 WT mice were purchased from Charles River. pIV−/− , Ciita−/4tgpIVKO , and Ragescribed . To seleLNSCs were obtained as previously described . In brieCells from LN, the spleen, the SC, and SGs were isolated by digesting organ fragments with an enzymatic mix containing collagenase D (1 μg/ml) and DNAse I (10 μg/ml) (Roche) in HBSS [14]. For SC and SG, single-cell suspensions were further centrifuged through a discontinuous 30:70% percoll (Invitrogen) gradient. T cells were further analysed by flow cytometry.Small intestines were excised and transferred into PBS 2% FCS. Peyer's patches and adipose tissue were carefully removed. The small intestines were cut longitudinally and cleaned of faecal content. The small intestines were next incubated in RPMI containing 10% FCS, 2 mM EDTA, and 1 mM DTT for 30 min at 37°C in a shaking incubator to remove epithelial cells, the supernatant being discarded. The remaining tissue was digested twice in RPMI containing 1 mg/ml of collagenase D (Roche) for 30 min at 37°C in a shaking incubator. Cells were recovered from the supernatant and filtered through a 70-μm cell strainer.d/I-Ed (2G9), anti-IFNγR (GR20), and H2-M (2E5A) were from BD. Anti-CD19 (1D3), anti-CD8 (53–6.7), anti-CD4 (GK1.5), anti-CD11b (M1/70), anti-CD62L (MEL-14), anti-PDCA-1 (eBio927), anti-PD-1 (J43), anti-IFN-γ (XMG1.2), anti-IL-17 (eBio17B7), and anti-Foxp3 (FJK-16s) were from eBioscience. Anti-EpCAM (caa7-9G8) and anti-Ly5.1 (6C3) were from Miltenyi Biotec.Anti-gp38 (8.1.1), anti-CD31 (390), anti-CD11c (N418), anti-CD44 (IM7), and anti-IAb (AF6.120.1) mAbs were from BioLegend. Anti-CD45 (30F11), anti-CD16/32 FcyRIII (2.4G2), anti-I-Aneg cells were stained with mAbs against CD45, gp38, and CD31. In some experiments, cells were also stained with mAbs against MHCII or isotype control as indicated. For cTEC and mTEC sorting, enriched CD45neg cells were stained with mAbs against CD45, EpCAM, and Ly5.1.For LNSC flow cytometry sorting, enriched CD452. Cells were stimulated for 18 h with PMA/ionomycin and Golgi stop solution (BD) was added during the last 4 h of culture before the staining.Intracellular cytokine stainings were carried out with the Cytofix/Cytoperm kit (BD) for IFN-γ and IL-17 staining. Foxp3 staining was performed with the eBioscience kit, according to manufacturer's instructions. For IFN-γ and IL-17 staining, LN cells were cultured in RPMI containing 10% heat-inactivated fetal bovine serum, 50 mM 2-mercaptoethanol, 100 mM sodium pyruvate, and 100 μM penicillin/streptomycin at 37°C and 5% COCells were either acquired on a Gallios or sorted using a MoFlowAstrios (Beckman Coulter), and analysed using FlowJo (Tree Star) or Kaluza softwares.Mice were transcardiacally perfused with PBS, and intestines were fixed in paraformaldehyde before inclusion in paraffin. Preserved organs were cut into 7-μm-thick sections and deparaffinised in xylene–ethanol. Ag retrieval was performed in citrate buffer. Sections were then stained using labelled antibodies against CD45 (30-F11) and CD3 (17A2) and DAPI (Sigma-Aldrich) counter-staining. Sections were mounted with Mowiol fluorescent mounting medium (EMD). Images were acquired with a confocal microscope .+Foxp3+) or non-proliferating (Foxp3+) cells, and the ratio of proliferating Tregs was calculated. At least 20 images/condition were quantified.Skin LNs from untreated mice or mice treated with IFN-γ and FTY720 were frozen in OCT medium. 10-μm-thick sections were cut and fixed with paraformaldehyde 4% for 20 min. After washing and permeabilization, the sections were stained overnight at 4°C using a rabbit anti-Lyve-1 antibody (Reliatech GmbH). Secondary staining was performed using a Alexafluor546-labelled donkey anti-rabbit antibody, Alexafluor488-labelled anti-Foxp3 (150D) antibodies and eF660-Ki67 (SolA15), for 2 h at room temperature. After DAPI (Sigma-Aldrich) staining, sections were mounted with Mowiol fluorescent mounting medium (EMD). Images were acquired with a confocal microscope . Tregs in “close proximity” (<5 μm) of LECs were quantified as proliferating (Ki67+ CD25hi T cells (Treg) were purified by flow cytometry from total skin LNs of 18-mo-old K14tgpIVKO and K14tg control mice and cultured at the indicated ratio with 2 × 105 proliferation dye-labeled naive CD4+ CD25neg T cells in the presence of bone marrow–derived DC and anti-CD3 antibodies. Labeled T-cell proliferation was assessed by flow cytometry after 5 d of co-culture.In vitro Treg suppressive assays were performed as follows: CD46 T cells were injected intravenously into Rag2−/− mice. Recipient mice were randomized before transfer and co-housed during the experiment.LN and spleen of donor mice were harvested, and T cells were negatively selected from the cell suspension using a PAN T isolation kit (Mylteniy biotec) according to the manufacturer's instructions. Purity generally exceeded 95%. 2–5 × 10Total RNA was isolated using Tri-Reagent (Ambien) from sorted cells. cDNA was synthesized using random hexamers and M-MLV reverse transcriptase (Promega). PCRs were performed with the CFX Connect real-time PCR detection system and iQ SYBR green super mix (Bio-Rad Laboratories). The results were normalized with GAPDH or 60S ribosomal protein L32 mRNA expression and quantified with a standard curve generated with serial dilutions of a reference cDNA preparation. Primer sequences: I-Ab forward, 5′-CTG TGG TGG TGG TGA TGG T-3′ and reverse, 5′-CGT TGG TGA AGT AGC ACT CG-3′. H2-Mα forward, 5′-CTCGAAGCATCTACACCAGTG-3′ and reverse, 5′-TCCGAGAGCCCTATGTTGGG-3′. K14 orward, 5′-AGG GAG AGG ACG CCC ACC TT-3′ and reverse, 5′- CCT TGG TGC GGA TCT GGC GG-3′K14tgpIVKO or control mice were visualized with HRP-conjugated goat anti-mouse IgG (Bio-Rad Laboratories) and the ECL WesternBright Sirius (advansta).Tissues were homogenized in T-PER tissue protein extraction reagent (Thermo Fisher Scientific) supplemented with a cocktail of protease inhibitors . Protein extracts were incubated at 95°C for 5 min with of 4× SDS–PAGE–loading buffer . Equal amounts of protein were run on 12% SDS–PAGE gels and transferred onto a polyvinylidene difluoride membrane . Sera from t test or two-way Anova, and Log-rank test using Prism software (GraphPad).Statistical significance was assessed by the two-tailed unpaired


Our results highlight the rapid response of DCs to Ad immunization and their role in mucosal immune activation and identify initial cellular mechanisms of the replicating Ad-SIV vaccine in the rhesus macaque model.Inducing strong mucosal immune responses by vaccination is important for providing protection against simian immunodeficiency virus (SIV). A replicating adenovirus type 5 host range mutant vector (Ad5hr) expressing SIV proteins induced mucosal immune responses in rectal tissue associated with delayed SIV acquisition in female rhesus macaques, but the initial mechanisms leading to the induced immunity have not been elucidated. As dendritic cells (DCs) are known to orchestrate both innate and adaptive effector immune cell responses, we investigated their role here. Rhesus macaques were immunized twice mucosally with a replicating Ad5hr expressing SIV Env, Gag, and Nef (Ad-SIV) or empty Ad5hr vector (Ad-Empty). DC subsets and their activation were examined in rectal tissue, blood, and LNs at 3 timepoints after each immunization. Plasmacytoid DCs, myeloid DCs, and Langerhans cells were significantly increased in the rectal mucosa, but only myeloid DCs were significantly increased in blood post-immunizations. All rectal DC subsets showed increased frequencies of cells expressing activation markers and cytokines post-immunization, blood DCs showed mixed results, and LN DCs showed few changes. Rectal DCs responded strongly to the vector rather than expressed SIV antigens, but rectal DC frequencies positively correlated with induced rectal antigen-specific memory T and B cells. These correlations were confirmed by  The  T cells , 6–9. Th+ and CD8+ T cell responses in local tissues and blood and antigen-specific T follicular helper cells in lymph nodes (LN) (While replication-incompetent adenoviruses (Ad) are being developed as HIV vaccine vectors –12, our des (LN) –20. Of rdes (LN) . A subsedes (LN) . However+ T cells. Subsequent antigen-specific antibody production by B cells and cytotoxic CD8+ T cell responses are dependent on the resultant CD4+ T cell help provided. DCs can also induce cytotoxic CD8+ T cell activation directly by cross-presentation. Since DCs are located within or beneath the epithelial lining of mucosal tissues, they are one of the first immune cells to encounter pathogens and immunogens -SIV recombinants express their inserted transgene at least 25 weeks. This expression persisted in mucosal macrophages and mDCs . HoweversmH4env, SIVgag, and SIVnef  at a dose of 5 × 108 plaque forming units/recombinant/site or with Ad5hr empty vector  at a dose equivalent to the Ad-SIV recombinants administered. All rhesus macaques were maintained at the NCI animal facility under the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. The protocols and procedures were approved by the NCI Animal Care and Use Committee. Immunization and sample collection schedules are shown in Female rhesus macaques were immunized at week 0  and week 12  with replicating Ad5hr recombinants separately expressing SIV+ cells. DCs were phenotyped by staining with BV711 anti-CD11c (3.9), Alexa Fluor 700 anti-HLA-DR (L243), Alexa Fluor 488 anti-CD14 (M5E2), and BV605 anti-CD1c (L161) (Biolegend) and BV412 anti-CD123 (7G3) (BD Biosciences). For analysis of DC activation markers, the cells were stained with the DC phenotyping antibodies plus PE-Cy5 anti-CD40 (5C3) and Biotin anti-CD86 (IT2.2) (BD Biosciences), PE-eFluor610 streptavidin, and PerCP-eFluor710 anti-CCR7 (3D12) (eBioscience). For DC cytokine production, the cells were incubated with BD Golgi-stop for 6 h and stained with DC surface phenotyping antibodies. Then the cells were stained with PE anti-interleukin (IL)-6 (MQ2-6A3) and PE-Cy7 anti-tumor necrosis factor (TNF)-α (MAB11) (BD Biosciences) and APC anti-B cell activating factor (BAFF) (1D6) (eBioscience) after fixation and permeabilization with a Cytofix/Cytoperm Kit (BD Biosciences). For intracellular cytokine staining of rectal T cells, cells obtained 21 days post-immunization were cultured with BD Golgi-stop, ECD anti-CD28 (CD28.2) (Beckman Coulter), purified CD49d (9F10) (eBioscience), and BV605 anti-CD4 (OKT4) (Biolegend). The cells were also stimulated with pooled peptides at 1 μg/ml final concentration representing SIVmac239 Gag (AIDS Research and Reference Reagent Program), SIVM766 gp120 Env  or SIVCG7V gp120 Env (ABL). The pools consisted of 15-mer peptides overlapping by 11 amino acids. After 6 h incubation, the cells were stained with Aqua Live/Dead viability dye, BV786 anti-CD3 (SP34.2) (BD Biosciences), eVolve655 anti-CD8 (RPA-T8), PE-CY5 anti-CD95 (DX2) (eBioscience), and then intracellularly stained following fixation/permeabilization with FITC anti-TNF-α (MAB11) (BD Bioscience), Alexa Fluor 700 anti-interferon (IFN)-γ (B27) (Life Technologies), and APC-Cy7 anti-IL-2 (MQ1-17H12) (Biolegend). For antigen-specific memory B cell staining, rectal cells obtained 21 days post-immunization were stained with biotinylated SIVCG7V, APC streptavidin, Alexa Fluor 700 anti-CD3 (SP34-2), BV650 anti-CD20 (2H7), BV605 anti-CD21 (B-ly4) (BD Biosciences), PE-Cy5 anti-CD19 (J3-119) (Beckman Coulter), and PE-TxRed anti-IgD (AT-1) (StemCell). The stained cells were acquired on an LSR II (BD Biosciences) and analyzed using FlowJo software.Rectal cells, PBMC, and LN cells were stained with Aqua Live/Dead viability dye (Invitrogen), V500 anti-CD3 (SP-34), and BV510 anti-CD56 (B159) (BD Biosciences), and BV510 anti-CD20 (2H7) (Biolegend) to exclude dead and lineage4 cells/well) and T cells (1 × 105 cells/well) were co-cultured in a U-bottom 96-well plate for 6 days. For the T cell proliferation assay, naïve T cells were stained with 3 μM of carboxyfluorescein succinimidyl ester  before co-culture and CFSElowCD3+CD4+ or CFSElowCD3+CD8+ cells were analyzed. The proliferated cell percentages are reported following subtraction of CFSElowCD3+CD4+ or CFSElowCD3+CD8+ values in co-cultures lacking DC. For cytokine assays, T cells were re-stimulated with or without Env peptide pools (SIVM766 plus SIVCG7V) at a 0.5 ug/ml final concentration plus BD Golgi-stop for 6 h after 6 days co-culture and then surface stained with Aqua Live/Dead viability dye, BV786 anti-CD3 (SP34.2), eVolve655 anti-CD8 (RPA-T8), and BV605 anti-CD4 (OKT4). Intracellular staining following fixation/permeabilization was then conducted as above for the rectal T cells. SIV Env-specific cytokine production of T cells was reported following subtraction of cytokine positive values in wells lacking peptide.Eight macaques from the Ad-SIV group and five macaques from the Ad-Empty group were used in co-culture experiments. Naïve autologous T cells were enriched using a pan T cell isolation kit (Miltenyi Biotech) from PBMC obtained prior to immunization . T and Bt-test. GraphPad Prism was used for statistical analysis.DC frequencies and activation in different groups at different times were analyzed by two-way ANOVA with the Tukey multiple-comparison test. Correlations were assessed using the Spearman rank correlation test and co-culture data were analyzed by unpaired + (pDC), CD11c+ (mDC), CD1ahighCD11c+ (LC), CD1c+CD11c+ (CD1c+ mDC), and CD1c−CD11c+ (CD1c− mDC) in the live lineage−CD14−HLA-DR+ population (− CD14−HLA-DR+ cell populations were significantly increased (2.4-fold in Ad-SIV and 2.5-fold in Ad-Empty groups) after the 2nd immunization  and tended to increase in the Ad-empty macaques  of CCR7+ pDCs and mDCs increased significantly following the 2nd immunization in the Ad-empty group, while the MFI of CD40+ pDCs and mDCs decreased  showed a positive correlation  , SIVM766 Env , and SIVCG7V Env . Rectal mDCs also correlated significantly with SIVM766-specific IFN-γ+CD4+ memory T cells  and SIVCG7V-specific memory B cells  , SIVM766 Env , and SIVCG7V Env , IFN-γ+CD4+ memory T cells specific for SIVM766 Env , and SIVCG7V-specific memory B cells  as well    and SIVCG7V-specific memory B cells   . Moreove 0.0054) . Among t as well . After t 0.0385) , the fre 0.0075) , and rec 0.0065) . As expe 0.0065) . These r−CD1c− cell purity and the enriched mDCs were 91.5% CD14−CD1c− and 6.39% CD14−CD1c+ (− suggested that the majority of antigen presentation and T cell activation in the co-cultures was attributable to DCs, and that any macrophage/monocyte effects were minimal. T cell proliferation and antigen-specific cytokine production after 6-days of co-culture were assessed. Without DCs, neither CD4+ nor CD8+ T cells proliferated (+ pDCs or CD11c+ mDCs stimulated autologous naïve T cell proliferation (+ T cells showed greater proliferation than CD4+ T cells, but there was no significant difference in proliferation between Ad-SIV and Ad-Empty groups or between pDCs and mDCs (+, IL-2+, and TNF-α+ CD4+ and CD8+ T cells, compared to co-culture with Ad-Empty DCs. CD8+ T cells from the Ad-SIV group tended to exhibit greater frequencies of cytokine+ cells, than CD4+ T cells, both individually and combined, but these differences were not significant. These data support the conclusion that the mucosal replicating Ad-SIV immunization elicited functional activation of rectal DCs with the potential to induce local and systemic antigen-specific immune responses.To evaluate rectal DC functions following immunization, we co-cultured rectal DCs with autologous naïve T cells. The naïve T cells were enriched from PBMCs which were collected before immunizations . DCs wer14−CD1c+ . CD14 isiferated . Howeverferation . CD8+ T and mDCs . Followiand mDCs . Co-cultAs most HIV infections occur via mucosal transmission, strong mucosal immune responses are desirable for an efficacious HIV vaccine , 37. In + rectal pDCs and mDCs increased indicating enhanced migration potential toward LNs (+ pDC frequencies in blood significantly increased (+, BAFF+, IL-6+, and TNF-α+ cells, but not CCR7+ cells after the 2nd Ad immunization (Newly generated immature DCs migrate from bone marrow to tissues via blood. Similarly, activated DCs in local tissues migrate to LNs via blood and lymphatics. Immature mucosal DCs are immobile but are activated once they capture foreign antigens, upregulate CCR7, and gain motility. CCR7 interacts with CCL21 on lymphatic endothelial cells, providing a path for DC migration into lymphatic vessels and draining LNs. Most DC migration is via lymphatics, but pDCs can gain access to blood through high endothelial venules by means of surface adhesion molecules such as L-selectin and P-selectin glycoprotein ligand 1 , 42. Herward LNs . In contncreased , supportnization . This bl+ populations (+ DCs. However, it's not clear why LN DCs did not show increased activation characteristics. The time of peak DC activation may have been earlier than 14 days post-immunization. In mice, once tissue-derived DCs arrive in draining LNs after activation, the survival time of mature DCs within the lymphoid organs is only 2–9 days (+ and CD86+ LN DCs before immunization were already higher than those in rectal and blood DCs. Due to the highly activated environment, detection of LN changes post-immunizations may have been difficult.In contrast to the significant DC recruitment and activation in rectal mucosa and blood, LN DCs did not show any changes in frequencies or activation marker expression post-immunizations except for the CCR7ulations ,7. Both 2–9 days , 44. MorMucosal DCs deliver and present antigenic information to naïve T cells in LNs, but they are also involved in T cell homing from the lymphoid organ to the mucosa after T cell activation. DCs from intestinal tissues induce expression of gut homing markers such as α4β7 and CCR9 on T and B cells , 45, 46.in vitro co-culture system, we demonstrated that rectal DCs were functionally activated after Ad immunizations and induced autologous naïve T cell activation (+ T cells were more proliferative than CD4+ T cells after co-culture with DCs, and there was no difference between pDC and mDC in inducing T cell proliferation. Because frequencies of activated rectal DCs from the Ad-Empty group were increased after immunization, it was not surprising that they induced some T cell proliferation. Nevertheless, after 6-days of co-culture and 6 h of Env peptide pool stimulation, T cells co-cultured with DCs from Ad-SIV-immunized macaques showed enhanced frequencies of cytokine+ cells approaching significance compared to those co-cultured with DCs from macaques that received Ad-Empty vectors (Using an tivation . CD8+ T  vectors . These rTaken together, our results show that mucosal Ad immunizations could recruit and activate DCs in rectal mucosa and blood and induce DC migration to draining lymph nodes. In addition, we showed that the activated mucosal DCs play a role in inducing antigen-specific T cell proliferation and cytokine production. Our findings identify the initial cellular mechanisms of the replicating Ad vaccine regimen in the rhesus macaque model and highlight the rapid response and potential roles of DCs in mucosal immune activation after replicating Ad immunizations. This information is valuable for development of HIV vaccines with enhanced efficacy against mucosal transmission and for improved mucosal vaccines in general.This study was carried out in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care. The protocol and procedures were approved by the National Cancer Institute Animal Care and Use Committee.E-JK and MR-G developed the concept and designed the experimental plans. E-JK, SH, GE-A, MR, and TH performed experiments. E-JK analyzed data. E-JK and MR-G prepared the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA-seq data presented in the article have been made publicly available on the NCBI Gene Expression Omnibus site (GEO), accession number GSE135833.Volume 7, issue 4, e00903-16, 2016,


Candida albicans infection can cause skin, vulvar, or oral pain. Despite the obvious algesic activity of C. albicans, the molecular mechanisms of fungal nociception remain largely unknown. Here we show that the C. albicans-specific signaling pathway led to severe mechanical allodynia. We discovered that C. albicans-derived β-glucan stimulated nociceptors depending on Dectin-1, and two pathways in inflammatory pain. The major pathway operates via the Dectin-1-mediated ATP-P2X3/P2X2/3 axis through intercellular relationships between keratinocytes and primary sensory neurons, which depends on the ATP transporter vesicular nucleotide transporter (VNUT). The other pathway operates via the Dectin-1-mediated PLC-TRPV1/TRPA1 axis in primary sensory neurons. Intriguingly, C. albicans-derived β-glucan has the ability to enhance histamine-independent pruritus, and VNUT inhibitor clodronate can be used to treat unpleasant feelings induced by β-glucan. Collectively, this is the first report to indicate that Dectin-1 and VNUT mediated innate sensory mechanisms that detect fungal infection.  •β-Glucan-induced pain is abolished in Dectin-1- and TRPV1/TRPA1-deficient mice•β-–Glucan stimulates nociceptors via Dectin-1-PLC axis, activating neurons•β-Glucan-induced allodynia is dependent on extracellular ATP and VNUT•VNUT inhibitor clodronate can be used to treat β-glucan-induced allodynia Molecular Mechanism of Behavior; Molecular Neuroscience; Medical Microbiology Candida albicans is an opportunistic fungus that thrives in the skin, mouth, vagina, and nipples. Immunodeficiency within hosts and poorly controlled diabetes have increased the rates of invasive C. albicans infections, evoking skin or oral pain. Notably, approximately 75% of women experience vulvovaginal candidiasis at some point during their lifetime and 5% of women experience recurrent episodes of infection . Notably, nociceptor-derived CGRP suppressed β-glucan-induced inflammation and osteoclast multinucleation via Jdp2-mediated NF-κB repression and inhibition of actin polymerization, respectively  induces robust pain via the Dectin-1-mediated ATP-P2X3/P2X2/3 axis and Dectin-1-mediated phospholipase C (PLC)-TRPV1/TRPA1 axis. Furthermore, CSBG has the ability to enhance Mrgpr ligand chloroquine (CQ)-induced itch behaviors. Strikingly, inhibition of the ATP transporter vesicular nucleotide transporter (VNUT) by using clodronate abolished the unpleasant feelings induced by β-glucan.In this study, we noticed that Dectin-1-deficient mice were unresponsive to fungal pain. C. albicans injected into the hind paw of mice induced pain-related behaviors  in the infected regions  analysis of resting-state brain fMRI -induced allodynia was the same between wild-type and Dectin-1-deficient mice  activation through P2X receptor in DRG neurons is involved in the pathogenesis of neuropathic pain  analysis depending on the red fluorescent protein (RFP) fluorescence level. Because the Dectin-1 expression level was higher in Nav1.8-positive neurons , we analyzed CSBG response in DRG neurons from wild-type and Dectin-1-deficient mice. Calcium imaging suggests the functional expression of Dectin-1 in isolated DRG neurons  with or without itching using a cheek injection model . Significantly, no allodynia was detected when CSBG was injected into the hind paw of VNUT-deficient mice, which cannot secrete ATP in response to Dectin-1 stimulation. A recent report suggested that clodronate inhibits VNUT at a half maximal inhibitory concentration of 15.6 nM without affecting other vesicular transporters, acting as an allosteric modulator through competition with Cl−  affects large numbers of women of childbearing age  deficient mice. To address such an important issue as future direction is important.All methods can be found in the accompanying


Furthermore, re-expression of miR-9 delays disease progression in EVI1high leukemia-xenograft mice. Our results suggest that EVI1-induced hypermethylation and downregulation of the miR-9 plays an important role in leukemogenesis in EVI-1high pediatric AML, indicating that hypomethylating agents may be a potential therapeutic strategy for EVI1high pediatric AML.Ectopic Viral Integration site 1 (EVI1) upregulation is implicated in 10–25% of pediatric acute myeloid leukemia (AML) and has an inferior outcome with current chemotherapy regimens. Here we report that EVI1 upregulation is associated with methylation of the miR-9 promoter and correlated with downregulation of miR-9 in human AML cell lines and bone marrow (BM) cells from pediatric patients. Reactivation of miR-9 by hypomethylating agents and forced expression of miR-9 in EVI1The online version of this article (10.1186/s12943-019-0952-z) contains supplementary material, which is available to authorized users. EVI1rognosis . Multiplrognosis .MicroRNA-9 (miR-9) is deregulated in several types of solid tumors serving as an oncogene in some and tumor suppressor in others . Low miRhigh cell lines (AML-1 and Kasumi-3) had significantly more methylation (85 and 70% respectively) versus EVI1low cell line (U937) 28% , which is a hypomethylating agent [high cell lines, AML-1 and Kasumi-3 compared to the EVI1low cell line U937  while all xenograft mice expressing miR-9 survived , indicating that ectopic expression of miR-9 delayed the disease latency in AML1 xenograft mice Additional file 2:Supplementary methods. (DOCX 19 kb)


SLC35B4 gene does not affect major UDP-Xyl- and UDP-GlcNAc-dependent glycosylation pathways.SLC35B4 belongs to the solute carrier 35 (SLC35) family whose best-characterized members display a nucleotide sugar transporting activity. Using an experimental model of HepG2 cells and indirect immunofluorescent staining, we verified that SLC35B4 was localized to the endoplasmic reticulum (ER). We demonstrated that dilysine motif, especially lysine at position 329, is crucial for the ER localization of this protein in human cells and therefore one should use protein C-tagging with caution. To verify the importance of the protein in glycoconjugates synthesis, we generated SLC35B4-deficient HepG2 cell line using CRISPR-Cas9 approach. Our data showed that knock-out of the  Glycosylation belongs to one of the most frequent posttranslational modifications of macromolecules. Synthesis and modification of glycan moiety is performed by glycosyltransferases located in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Glycosyltransferases use sugars activated by the addition of a nucleoside mono- or diphosphate  as substrates. Nucleotide sugars are synthesized in the cytosol , and in SLC35B4 gene is mapped to chromosome 7q33 and encodes two splice variants: a longer version (encoding a protein of 331 amino acids) [et al. [Saccharomyces cerevisiae cells expressing the longer splice variant of the human SLC35B4 protein showed specific uptake of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-xylose (UDP-Xyl). Roy et al. [S. cerevisiae SLC35B4 homolog (YEA4) transported UDP-GlcNAc. Microsomes derived from V79 cells (Chinese hamster lung fibroblasts) overexpressing both splice variants of the SLC35B4 transporter showed specific uptake of UDP-glucuronic acid (UDP-GlcA), but only after preloading of microsomes with UDP-GlcNAc [et al. [Drosophila melanogaster SLC35B4/YEA4 homolog (Efr) also transported GDP-fucose (GDP-Fuc).The first characterized NSTs were demonstrated to be specific for the translocation of a single nucleotide sugar ,7,14, buo acids) ,18 and a [et al. , vesicley et al.  demonstrP-GlcNAc . Ishikaw [et al.  found th2) in Minimum Essential Medium Eagle . Medium was supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Cytogen). Cells were stably transfected with the respective plasmids using FuGENE 6 transfection reagent (Promega) according to the manufacturer’s protocol. Stable transfectants overexpressing SLC35B4 tagged with HA at the C-terminus, N-terminus or modified variants of the recombinant N-tagged protein were selected in complete media containing 200 μg/ml zeocin (InvivoGen). Cells were analyzed for overexpression of HA-tagged proteins using indirect immunofluorescent staining.HepG2 cells  were maintained under humidified atmosphere (37°C and 5% COSLC35B4 gene using QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies) and primers listed in Total RNA was isolated from HeLa cells, and cDNA encoding human SLC35B4 protein (NCBI accession number AAH08413) was synthesized as described previously . PCR proHepG2 cells were transfected with a mixture of human SLC35B4 double nickase plasmids according to the manufacturer's instructions (Santa Cruz Biotechnology) and selection was performed in MEM complete medium supplemented with 1 μg/ml puromycin (InvivoGen). Twelve independent clones overexpressing GFP (the second selection marker in the utilized CRISPR-Cas9 system) were isolated. To verify gene knock-out, total RNA and genomic DNA were isolated from cells and RT-PCR and PCR reactions were performed, respectively, using HepG2 wild-type cells as a control. To identify mutations introduced by the CRISPR-Cas9 approach, PCR products obtained using cDNA as a template were cloned into the pJET1.2/blunt vector according to the manufacturer's instructions (Thermo Fisher Scientific) and sequenced using pJET1.2 forward and pJET1.2 reverse sequencing primers (Thermo Fisher Scientific). PCR products obtained from genomic DNA as a template were sequenced directly without cloning using hsSLC35B4_DNA_F and hsSLC35B4_DNA_R primers .Cells were seeded onto glass 8-well microscope slides (Merck) coated with 0.01% poly-L-lysine (Sigma-Aldrich), fixed and subsequently treated as described in details previously . BrieflyN-glycans, permethylation of isolated oligosaccharides without desialylation was carried out [O-glycans, modified procedure by Kudelka et al. [5) were seeded onto 10-cm dishes  in 10 ml of a complete growth medium containing 10% FBS. After 24 hours medium was replaced with 10 ml of medium with reduced (5%) FBS content, supplemented with Ac3GalNAcBn (50 μM) and cells were grown for additional 72 hours. Next, conditioned media were collected, centrifuged (1000×g) in order to remove cells detached upon medium aspiration and the resulting supernatants were subjected to glycan extraction procedure [To analyze ried out ,24 and dFor immunodetection of selected proteoglycans, cell lysates were subjected to SDS-PAGE using 8% polyacrylamide gels and after transferring onto nitrocellulose membranes visualized with mouse anti-chondroitin-4-sulfate  and mouse anti-keratan sulfate  antibodies followed by anti-mouse IgG antibody conjugated with HRP . For detection of chondroitin-4-sulfate, before SDS-PAGE cell lysates were treated with 0.2 U/ml chondroitinase ABC (Sigma Aldrich) in 50 mM Tris-HCl, containing 60 mM sodium acetate for 2 h at 37°C. Protein loading was assessed using Coomassie Brilliant Blue G-250 (CBB) staining.2O or 50% MeOH depending on their solubility. Glycan solution (5 μl) was combined with matrix (5 μl) and the resulting mixture (1 μl) was spotted on the MALDI plate and allowed to air dry. As a matrix, 20 mg/ml 2,5-dihydroxybenzoic acid (Sigma-Aldrich) dissolved in 20 mM sodium acetate buffer, 20% MeCN was used. Mass spectrum was accumulated from at least 100 laser shots and processed by Data Explorer ver. 4 (Applied Biosystems) and Microsoft Office 2013.Positive ion MALDI mass spectra were recorded on the Voyager-Elite (PerSeptive Biosystems) instrument equipped with nitrogen laser (337 nm) in reflector mode at an acceleration voltage of 20 kV . Vacuum-dried glycan samples were dissolved in 10–20 μl of either HS. cerevisiae (YEA4) and D. melanogaster (Efr) have been found in the ER of yeasts and fly epithelium of third instar wing discs, respectively [Ricinus communis agglutinin (MDCK-RCAr) [SLC35B4 gene) stably overexpressing HA-tagged protein and indirect immunofluorescent imaging. We have chosen this cell line because our preliminary studies showed that mRNA encoding SLC35B4 is expressed at different levels in cell lines and human tissues, with higher levels in liver tissue among others , localized to the ER [Literature data has shown that transiently overexpressed human SLC35B4 localized in CHO-K1 cells to the Golgi apparatus . In contectively ,20. Our CK-RCAr) . These c others  has been suggested to transport GDP-Fuc [O-fucosylation of Notch [et al. [et al. [Drosophila Efr, respectively, both proteins are homologous proteins produced by different organisms and therefore they may differ in biological activity as compared to mammalian SLC35B4. It has been reported that the expression of the human SLC35B4 gene is relatively high in the liver , deletions of 270-bp in three knock-out (KO) clones are shown as dashed lines (—), sequences used to design primers and amplify the genomic fragment are marked in gray, sequences used to design guide RNAs for CRISPR-Cas knock-out plasmids are shown in bold and underlined.Exon #1 of (DOCX)Click here for additional data file.


PMF BM-derived CD14+/CD34- monocytes engrafted and induced a PMF-like phenotype with splenomegaly, myeloid hyperplasia with clusters of atypical megakaryocytes, persistence of the JAK2V617F mutation, and BM and spleen fibrosis. As control we used normal human BM-derived CD14+/CD34- monocytes. These monocytes also engrafted and gave rise to normal megakaryocytes that, like PMF CD14+/CD34--derived megakaryocytes, expressed HLA-ABC and human CD42b antigens. Using 2 clonogenic assays we confirmed that PMF and normal BM-derived CD14+/CD34- monocytes give rise to megakaryocyte colony-forming cells, suggesting that a subpopulation BM monocytes harbors megakaryocyte progenitor capacity. Taken together, our data suggest that PMF monocytes induce myelofibrosis-like phenotype in immunodeficient mice and that PMF and normal BM-derived CD14+/CD34- monocytes give rise to megakaryocyte progenitor cells.To confirm that neoplastic monocyte-derived collagen- and fibronectin-producing fibrocytes induce bone marrow (BM) fibrosis in primary myelofibrosis (PMF), we injected PMF BM-derived fibrocyte-precursor CD14 Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by progressive bone marrow (BM) fibrosis., 2 In apChallenging the dogma that BM fibrosis is induced by mesenchymal stromal cells (MSC) overstimulated by cytokines produced by megakaryocytes and platelets,, 7 we fo+/CD34- monocytes engrafted in NSG mice and induced BM fibrosis. Similarly, normal BM CD14+/CD34- cells engrafted in NSG mice and, like PMF BM CD14+/CD34- cells, gave rise to human megakaryocytes both in vivo and in vitro.Because we found that PMF BM low-density cells engrafted in NOD-SCID-γ (NSG) mice and induced a PMF-like phenotype, we wonde+/CD34- cells using anti-CD14(PerCP-CY5.5) and anti-CD34 (FITC) antibodies.Diagnostic BM specimens were obtained from treatment-naïve PMF patients after an MD Anderson Cancer Center Institutional Review Board (MDACC IRB1) approved written informed consent had been obtained. The patients’ clinical data are provided in +/CD34- cell population was assessed by flow cytometry (FCM) using anti-CD14 (PerCP) and anti-CD34 (FITC) antibodies. In addition, PMF patients’ and healthy donors’ BM aspirates were further analyzed using antibodies or following 9 cell surface antigens: CD14 (PerCP-Cy5.5), CD34 (FITC), CD45 (Pacific Orange), CD68 (PE), CD3 (APC), HLA-DR (APC-H7), CD41 (PE/Cy7), CD42b (Alexa Fluor 700), and CD61 (BV421) using the FlowJo software v10.5.3  and R-based pipeline for high-dimensional cytometry datasets, as previously described.[+ cells were gated and the expression of each of the above mentioned surface markers was assessed. The antibodies corresponding isotypes were used as controls. In addition, cell surface marker intensities of 4,000 randomly selected cells were obtained in each sample and mapped to a comparable range using arcsinh transformation with a cofactor of 150. Clusters were computationally defined using the unsupervised self-organizing map (SOM) clustering in the FlowSOM package within the R environment . The antibodies and their isotype controls used in our study are listed in The purity of the fractionated CD14escribed., 12 Briescid gamma mice  were included in this study. All animal studies were carried out in strict accordance to an animal protocol approved by the MD Anderson Cancer Center Instructional Animal Care and Utilization Committee (Protocol Number: 00000787-RN02). The animals were euthanized using carbon dioxide as per our protocol and all efforts were made to minimize suffering. Two million PMF or hematologically normal BM-derived CD14+/CD34- cells were injected into the tail vein of 4–6 week old female NOD scid gamma mice. Engraftment was assessed after two weeks and then once a month thereafter by FCM of mouse peripheral blood (PB) cells using mouse anti-human leukocyte antigen (HLA)-ABC antibody and by allele-specific suppressive quantitative polymerase chain reaction (qASSPCR) analyses for the quantification of the JAK2V617F exon 14 mutation.[A total of 30 NOD mutation. FollowinFemur, sternum and spleen biopsy samples were taken after the animals were euthanized . BM and spleen biopsy morphological analyses were performed using hematoxylin and eosin (H&E) and silver staining as previously described. The degr+/CD34- cells (n = 6) was assessed by using two different megakaryocyte colony culture assays. The collagen-based MegaCult-C assay  was used in accordance with the manufacturer’s instructions with slight modifications. In brief, 40,000 CD14+/CD34- cells were cultured in collagen-containing medium in triplicate in the presence of recombinant human thrombopoietin , interleukin-3  and IL-6 (10 ng/mL). Colony-forming unit-megakaryocyte (CFU-Meg) colonies were fixed after 12 days of culture, stained with anti-CD42b (Vector Blue) antibodies, counterstained with nuclear fast red, and counted using BX43 upright brightfield microscope . A CFU-Meg colony was defined as a cluster of 3 or more nucleated CD42b+ cells. In addition, to further assess antigen co-localization, cells were stained with fluorescent actin , CD42b (Alexa Fluor 594) and CD14 (Alexa Fluor 647) antibodies with 4′,6-diamidino-2-phenylindole (DAPI) as the nuclear counterstain.The megakaryocyte colony-forming capacity of PMF (n = 12) and normal BM-derived CD145 PMF or normal BM-derived CD14+ cells were mixed with 0.8% methylcellulose in Iscove’s modified Dulbecco’s medium  supplemented with 10% fetal calf serum (Invitrogen) and 10 ng/mL thrombopoietin. Five-hundred mL of the mix was incubated in Petri dishes at 37°C in a humidified atmosphere of 5% CO2 in air. CFU-Meg colonies were scored at day 21 of culture using the Diaphot ELWD 0.3 inverted phase-contrast microscope . A CFU-Meg colony was defined as a cluster of more than 10 large translucent cells or adjacent clusters or 3 or more translucent cells. Single colonies were microaspirated, cytospun onto glass slides and analyzed after May-Grünwald-Giemsa (MGG) staining and immunolabeling with anti-CD42b (DAB) antibodies as described above. To assess differences between colony numbers, the Prism v7.03 software  was used. Groups were compared using Student’s t-test and P < 0.05 was considered statistically significant. Fluorescence images were acquired using the Andor Revolution XDi WD spinning disk confocal system with Zyla 4.2 sCMOS camera  and UPlanSApo 30XS silicone oil-immersion objective lens , and adjusted for visual presentation using Imaris v9.1.0 software . Antibodies and detection reagents used in the cellular imaging are listed in A previously described, slightly modified methylcellulose CFU-Meg colony culture assay was also used. Briefly,+ cells do not express the CD34 antigen (+ BM monocytes from PMF patients’ BM aspirates (n = 3) and, as control, from BM aspirates of healthy individuals (n = 2). Of the CD14+ BM cells, 97.6 ± 1.0% were CD14+/CD34- . Although we observed a decline in the JAK2V617F allele burden at the time of final bleed, the human mutant JAK2 variant was still detectable at a significant level several months after transplantation  or normal BM CD14+ cells (n = 2), using megakaryocyte , and fibrocyte  markers  of CD14+ monocytes expresses megakaryocyte lineage surface markers. To answer this question we performed a high-parametric immunophenotype analysis of PMF and normal BM CD14+ cells. We found that the vast majority of CD14+ cells co-express CD45 and HLA-DR, but not CD34, CD68 or CD3 antigens  that expresses all 3 megakaryocyte markers Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.