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Document describing important mouse lines, treatment protocols, antibodies and primers used in the present study.

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations.Significance:This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAFV600E genetic alteration is found in 60% of this endocrine cancer. This oncogene is associated with poor prognosis, resistance to radioiodine therapy, and tumor progression. Histological follow-up by anatomo-pathologists revealed that two-thirds of surgically-removed thyroids do not present malignant lesions. Thus, continued fundamental research into the molecular mechanisms of TC downstream of BRAFV600E remains central to better understanding the clinical behavior of these tumors. To study PTC, we used a mouse model in which expression of BRAFV600E was specifically switched on in thyrocytes by doxycycline administration. Upon daily intraperitoneal doxycycline injection, thyroid tissue rapidly acquired histological features mimicking human PTC. Transcriptomic analysis revealed major changes in immune signaling pathways upon BRAFV600E induction. Multiplex immunofluorescence confirmed the abundant recruitment of macrophages, among which a population of LYVE-1+/CD206+/STABILIN-1+ was dramatically increased. By genetically inactivating the gene coding for the scavenger receptor STABILIN-1, we showed an increase of CD8+ T cells in this in situ BRAFV600E-dependent TC. Lastly, we demonstrated the presence of CD206+/STABILIN-1+ macrophages in human thyroid pathologies. Altogether, we revealed the recruitment of immunosuppressive STABILIN-1 macrophages in a PTC mouse model and the interest to further study this macrophage subpopulation in human thyroid tissues.

Papillary thyroid carcinoma (PTC) is the most frequent histological subtype of thyroid cancers (TC), and BRAF

Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAF

BACKGROUND AND AIMS: Earlier diagnosis and treatment of intrahepatic cholangiocarcinoma (iCCA) are necessary to improve therapy, yet limited information is available about initiation and evolution of iCCA precursor lesions. Therefore, there is a need to identify mechanisms driving formation of precancerous lesions and their progression toward invasive tumors using experimental models that faithfully recapitulate human tumorigenesis. APPROACH AND RESULTS: To this end, we generated a mouse model which combines cholangiocyte-specific expression of KrasG12D with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced inflammation to mimic iCCA development in patients with cholangitis. Histological and transcriptomic analyses of the mouse precursor lesions and iCCA were performed and compared with human analyses. The function of genes overexpressed during tumorigenesis was investigated in human cell lines. We found that mice expressing KrasG12D in cholangiocytes and fed a DDC diet developed cholangitis, ductular proliferations, intraductal papillary neoplasms of bile ducts (IPNBs), and, eventually, iCCAs. The histology of mouse and human IPNBs was similar, and mouse iCCAs displayed histological characteristics of human mucin-producing, large-duct-type iCCA. Signaling pathways activated in human iCCA were also activated in mice. The identification of transition zones between IPNB and iCCA on tissue sections, combined with RNA-sequencing analyses of the lesions supported that iCCAs derive from IPNBs. We further provide evidence that tensin-4 (TNS4), which is stimulated by KRASG12D and SRY-related HMG box transcription factor 17, promotes tumor progression. CONCLUSIONS: We developed a mouse model that faithfully recapitulates human iCCA tumorigenesis and identified a gene cascade which involves TNS4 and promotes tumor progression. ispartof: HEPATOLOGY vol:74 issue:3 pages:1445-1460 ispartof: location:United States status: published

Selenium is an underexplored element that can be used for bioisosteric replacement of lower molecular weight chalcogens such as oxygen and sulfur. More studies regarding the impact of selenium substitution in different chemical scaffolds are needed to fully grasp this element's potential. Herein, we decided to evaluate the impact of selenium incorporation in a series of tryptophan 2,3-dioxygenase (TDO2) inhibitors, a target of interest in cancer immunotherapy. First, we synthesized the different chalcogen isosteres through Suzuki-Miyaura type coupling. Next, we evaluated the isosteres' affinity and selectivity for TDO2, as well as their lipophilicity, microsomal stability and cellular toxicity on TDO2-expressing cell lines. Overall, chalcogen isosteric replacements did not disturb the on-target activity but allowed for a modulation of the compounds' lipophilicity, toxicity and stability profiles. The present work contributes to our understanding of oxygen/sulfur/selenium isostery towards increasing structural options in medicinal chemistry for the development of novel and distinctive drug candidates.

Exacerbated IL-22 activity induces tissue inflammation and immune disorders such as psoriasis. However, because IL-22 is also essential for tissue repair and defense at barrier interfaces, targeting IL-22 activity to treat psoriasis bears the risk of deleterious effects at mucosal sites such as the gut. We previously showed in vitro that IL-22 signaling relies on IL-22 receptor alpha (IL-22Rα) Y-dependent and -independent pathways. The second depends on the C-terminal Y-less region of IL-22Rα and leads to a massive signal transducer and activator of transcription 3 (STAT3) activation. Because STAT3 activation is associated with the development of psoriasis, we hypothesized that the specific inhibition of the noncanonical STAT3 activation by the Y-less region of IL-22Rα could reduce psoriasis-like disease while leaving intact its tissue defense functions in the gut. We show that mice expressing a C-terminally truncated version of IL-22Rα (ΔCtermut/mut mice) are protected from the development of psoriasis-like dermatitis lesions induced by imiquimod to a lesser extent than Il22ra–/– mice. In contrast, only Il22ra–/– mice lose weight after Citrobacter rodentium infection. Altogether, our data suggest that specific targeting of the noncanonical STAT3 activation by IL-22 could serve to treat psoriasis-like skin inflammation without affecting IL-22‒dependent tissue repair or barrier defense at other sites.

Pancreatic acinar cells are a cell type of origin for pancreatic cancer that become progressively less sensitive to tumorigenesis induced by oncogenic Kras mutations after birth. This sensitivity is increased when Kras mutations are combined with pancreatitis. Molecular mechanisms underlying these observations are still largely unknown. To identify these mechanisms, we generated the first CRISPR-edited mouse models that enable detection of wild-type and mutant KRAS proteins in vivo. Analysis of these mouse models revealed that more than 75% of adult acinar cells are devoid of detectable KRAS protein. In the 25% of acinar cells expressing KRAS protein, transcriptomic analysis highlighted a slight upregulation of the RAS and MAPK pathways. However, at the protein level, only marginal pancreatic expression of essential KRAS effectors, including C-RAF, was observed. The expression of KRAS and its effectors gradually decreased after birth. The low sensitivity of adult acinar cells to Kras mutations resulted from low expression of KRAS and its effectors and the subsequent lack of activation of RAS/MAPK pathways. Pancreatitis triggered expression of KRAS and its effectors as well as subsequent activation of downstream signaling; this induction required the activity of EGFR. Finally, expression of C-RAF in adult pancreas was required for pancreatic tumorigenesis. In conclusion, our study reveals that control of the expression of KRAS and its effectors regulates the sensitivity of acinar cells to transformation by oncogenic Kras mutations. Significance: This study generates new mouse models to study regulation of KRAS during pancreatic tumorigenesis and highlights a novel mechanism through which pancreatitis sensitizes acinar cells to Kras mutations.

The shedding of extracellular vesicles (EVs) from the red blood cell (RBC) surface is observed during senescence in vivo and RBC storage in vitro. Two main models for EV shedding, respectively based on calcium rise and oxidative stress, have been proposed in the literature but the role of the plasma membrane lipid composition and properties is not understood. Using blood in K+/EDTA tubes stored for up to 4 weeks at 4°C as a relevant RBC vesiculation model, we showed here that the RBC plasma membrane lipid composition, organization in domains and biophysical properties were progressively modified during storage and contributed to the RBC vesiculation. First, the membrane content in cholesterol and linoleic acid decreased whereas lipid peroxidation and spectrin:membrane occupancy increased, all compatible with higher membrane rigidity. Second, phosphatidylserine surface exposure showed a first rapid rise due to membrane cholesterol decrease, followed by a second calcium-dependent increase. Third, lipid domains mainly enriched in GM1 or sphingomyelin strongly increased from the 1st week while those mainly enriched in cholesterol or ceramide decreased during the 1st and 4th week, respectively. Fourth, the plasmatic acid sphingomyelinase activity considerably increased upon storage following the sphingomyelin-enriched domain rise and potentially inducing the loss of ceramide-enriched domains. Fifth, in support of the shedding of cholesterol- and ceramide-enriched domains from the RBC surface, the number of cholesterol-enriched domains lost and the abundance of EVs released during the 1st week perfectly matched. Moreover, RBC-derived EVs were enriched in ceramide at the 4th week but depleted in sphingomyelin. Then, using K+/EDTA tubes supplemented with glucose to longer preserve the ATP content, we better defined the sequence of events. Altogether, we showed that EV shedding from lipid domains only represents part of the global vesiculation mechanistics, for which we propose four successive events (cholesterol domain decrease, oxidative stress, sphingomyelin/sphingomyelinase/ceramide/calcium alteration and phosphatidylserine exposure).

Expression of the chemokine receptor Ccr6 is shared by most IL-22–producing cells, and Ccr6-deficient mice showed decreased IL-22 production and skin inflammation upon IL-23 intradermal injections. To determine whether this observation might be extended to another psoriasis model, we applied imiquimod on Ccr6-deficient mice. Although epidermal IL-22 production was decreased because of a deficient recruitment of γδ T cells in these mice, they were not protected against psoriatic lesions. When primary epidermis or dermis tissue culture cells from nontreated mice were stimulated ex vivo with IL-1α/IL-2/IL-23, we observed that Ccr6 is crucial for Il22 expression from epidermal but not dermal cultures. Taking advantage of Ccr6-LacZ–knock-in mice, we showed that Ccr6 is necessary for the homing of Ccr6-positive cells, probably a γδ T-cell subset, which represents the main potential IL-22 source in the epidermis. Similar results were observed in Rag1 –/– epidermis and dermis primary cultures, in which a subset of innate lymphoid cells expressing Ccr6 represents the main potential source of IL-22. Taken together, our data show that Ccr6 is not required for the development of skin lesions induced by imiquimod despite its effect on epidermal homing of IL-22–producing cells.

Introduction Viruses have developed multiple mechanisms to alter immune reactions. In 1969, it was reported that lactate dehydrogenase-elevating virus (LDV), a single stranded positive sense mouse nidovirus, delays skin allograft rejection and inhibits spleen alterations in graft versus host disease (GVHD). As the underlying mechanisms have remained unresolved and given the need for new therapies of this disease, we reassessed the effects of the virus on GVHD and tried to uncover its mode of action. Methods GVHD was induced by transfer of parent (B6) spleen cells to non-infected or LDV-infected B6D2F1 recipients. In vitro mixed-lymhocyte culture (MLC) reactions were used to test the effects of the virus on antigen-presenting cells (APC) and responder T cells. Results LDV infection resulted in a threefold increase in survival rate with reduced weight loss and liver inflammation but with the establishment of permanent chimerism that correlated with decreased interleukine (IL)-27 and interferon (IFN)γ plasma levels. Infected mice showed a transient elimination of splenic CD11b+ and CD8α+ conventional dendritic cells (cDCs) required for allogeneic CD4 and CD8 T cell responses in vitro. This drop of APC numbers was not observed with APCs derived from toll-like receptor (TLR)7-deficient mice. A second effect of the virus was a decreased T cell proliferation and IFNγ production during MLC without detectable changes in Foxp3+ regulatory T cell (Tregs) numbers. Both cDC and responder T cell inhibition were type I IFN dependent. Although the suppressive effects were very transient, the GVHD inhibition was long-lasting. Conclusion A type I IFN-dependent suppression of DC and T cells just after donor spleen cell transplantation induces permanent chimerism and donor cell implantation in a parent to F1 spleen cell transplantation model. If this procedure can be extended to full allogeneic bone marrow transplantation, it could open new therapeutic perspectives for hematopoietic stem cell transplantation (HSCT).

In the developing spinal cord, Onecut transcription factors control the diversification of motor neurons into distinct neuronal subsets by ensuring the maintenance of Isl1 expression during differentiation. However, other genes downstream of the Onecut proteins and involved in motor neuron diversification have remained unidentified. In the present study, we generated conditional mutant embryos carrying specific inactivation of Onecut genes in the developing motor neurons, performed RNA-sequencing to identify factors downstream of Onecut proteins in this neuron population, and employed additional transgenic mouse models to assess the role of one specific Onecut-downstream target, the transcription factor Nkx6.2. Nkx6.2 expression was up-regulated in Onecut-deficient motor neurons, but strongly downregulated in Onecut-deficient V2a interneurons, indicating an opposite regulation of Nkx6.2 by Onecut factors in distinct spinal neuron populations. Nkx6.2-null embryos, neonates and adult mice exhibited alterations of locomotor pattern and spinal locomotor network activity, likely resulting from defective survival of a subset of limb-innervating motor neurons and abnormal migration of V2a interneurons. Taken together, our results indicate that Nkx6.2 regulates the development of spinal neuronal populations and the formation of the spinal locomotor circuits downstream of the Onecut transcription factors.

Background & AimsAlthough earlier diagnosis and treatment of intrahepatic cholangiocarcinoma (iCCA) is necessary to improve therapy, there is still limited information available about initiation and evolution of iCCA precursor lesions. Therefore, there is a need to identify mechanisms driving formation of precancerous lesions and their progression towards invasive tumor using experimental models that faithfully recapitulate human tumorigenesis.MethodsWe generated a new mouse model which combines cholangiocyte-specific expression ofKrasG12Dwith 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-induced inflammation to mimic iCCA development in patients with cholangitis. Histological and transcriptomic analyses of the mouse precursor lesions and iCCA were performed and compared with human analyses. The function of genes overexpressed during tumorigenesis was investigated in human cell lines.ResultsMice expressingKrasG12Din cholangiocytes and fed a DDC diet developed cholangitis, ductular proliferations, intraductal papillary neoplasms of bile ducts (IPNBs) and eventually iCCAs. The histology of mouse and human IPNBs were highly similar, and mouse iCCAs displayed histological characteristics of human mucin-producing large duct type iCCA. Signaling pathways activated in human iCCA were activated in mice. The identification of transition zones between IPNB and iCCA on tissue sections, combined with RNA-sequencing analyses of the lesions supported that iCCAs derive from IPNBs. We provide evidence that a gene cascade which comprisesKRASG12D, SRY-related HMG box transcription factor 17 (SOX17) and Tensin 4 (TNS4), and which is activated by epidermal growth factor, promotes tumor progression.ConclusionsWe developed a novel mouse model that faithfully recapitulates human iCCA tumorigenesis and identified a gene cascade promoting tumor progression.

Different studies on AGuIX nanoparticles have been achieved in the biomedical domain, showing that they allow us to combine multimodal and theranostic properties in oncology. The targeting of apoptotic cells presents a wide range of biomedical applications, including the monitoring of antitumoral therapy and the diagnosis of diseases related to this process, such as atherosclerosis, ischemia, chronic inflammation or autoimmune disorders. AGuIX nanoparticles functionalized with a peptide that recognizes apoptotic cells and with organic fluorophores were characterized by several physicochemical and biological methods such as HPLC, relaxometry and photon correlation spectroscopy, which attested to their potential as bimodal tracers detected by optical imaging and MRI. An increase of relaxivity and stability of AGuIX nanoparticles is also observed after their vectorization. The biological efficiency of this novel bimodal probe to target apoptotic cells was evaluated by fluorescence microscopy, relaxometry, MRI and flow cytometry on a lymphoblastic human T-cell line. In vitro cell apoptosis was chemically induced by incubation with camptothecin. Our in vitro experiments showed a significant specificity of vectorized AGuIX nanoparticles for camptothecin-treated cells that suggests their potential efficiency as probes to target apoptosis. Copyright © 2016 John Wiley & Sons, Ltd.

IL-22 has a detrimental role in skin inflammatory processes, for example in psoriasis. As transcription factor, AhR controls the IL-22 production by several cell types (i.e. Th17 cells). Here, we analyzed the role of Ahr in IL-22 production by immune cells in the inflamed skin, using an imiquimod-induced psoriasis mouse model. Our results indicate that IL-22 is expressed in the ear of imiquimod-treated Ahr(-/-) mice but less than in wild-type mice. We then studied the role of AhR on three cell populations known to produce IL-22 in the skin: γδ T cells, Th17 cells, and ILC3, and a novel IL-22-producing cell type identified in this setting: CD4(-) CD8(-) TCRβ(+) T cells. We showed that AhR is required for IL-22 production by Th17, but not by the three other cell types, in the imiquimod-treated ears. Moreover, AhR has a role in the recruitment of γδ T cells, ILC3, and CD4(-) CD8(-) TCRβ(+) T cells into the inflamed skin or in their local proliferation. Taken together, AhR has a direct role in IL-22 production by Th17 cells in the mouse ear skin, but not by γδ T cells, CD4(-) CD8(-) TCRβ(+) T cells and ILCs.

Kras mutations are not sufficient to induce precancerous lesions in the pancreas, unlike in other organs such as lungs. Pancreatic intraepithelial neoplasia (PanIN) appear when mutated Kras is associated with pancreatitis. Since acinar cells are thought to be the cell origin of PanIN and pancreatic ductal adenocarcinoma (PDAC), our aim is to understand how acinar cells initially resist to Kras mutations and how pancreatitis enables mutated Kras to promote pancreatic neoplasia. Pancreatitis and associated inflammation were induced by injections of cerulein in tamoxifen-treated Elastase-CreER/LSLKrasG12D mice, in which mutated Kras expression is induced exclusively in acinar cells. RNAseq analysis on FACS-sorted acinar cells revealed that Kras expression and several cancer-associated pathways were rapidly induced in the presence of inflammation. In situ hybridization, RTqPCR, and Western blot (WB) experiments confirmed that Kras and/or KrasG12D mRNA and protein were significantly increased in acinar cells after cerulein treatment. To further explore the control of Kras expression, we generated a novel mouse model in which a citrine gene (a variant of GFP) is fused in frame with the endogenous Kras gene, allowing to detect Kras on tissue sections using a GFP antibody. We demonstrated by WB that the protein expression of Kras, KrasG12D and citrine-Kras fusion was similarly regulated in response to cerulein treatment, suggesting the involvement of the same regulatory elements on all three alleles. The ability of citrine-Kras fusion to bind GTP and translocate to the plasma membrane in acini was not altered compared to wild-type Kras protein. Little or no citrine-Kras was visualized in acinar cells of tamoxifen-treated Elastase-CreER/LSLKrasG12D/citrine-Kras mice. However, a 30-fold increase of citrine-Kras at the cell membrane of neoplastic lesions was detected after cerulein treatment. This indicates that inflammation is required for expression and plasma membrane targeting of Kras and KrasG12D in metaplastic acini. Importantly, protein expression of FT-α/β, Rce1 and ICMT, three enzymes necessary for Kras targeting to the membrane was greatly increased in murine acinar cells during pancreatitis. Basal FT-α/β, Rce1 and ICMT levels were found to be very low in normal mouse and human pancreata, compared to other organs, and their expression increased in primary mouse and human acini grown under conditions inducing the formation of preneoplastic lesions. In addition, computational analysis of a PDAC cohort (n=178) from The Cancer Genome Atlas (TCGA) revealed a positive correlation between expression of KrasG12D and its regulating enzymes. We conclude that Kras and KrasG12D are normally not present in acinar cells in vivo and that inflammation induces a coordinated program driving their expression and translocation to plasma membrane. Our findings provide a first clue to the long-standing observation that in several organs, Kras mutations, alone, are not sufficient to affect tissue homeostasis. Citation Format: Mohamad Nabil Assi, Jean-Nicolas Lodewyckx, Younes Achouri, Claude Gérard, Nicolas Dauguet, Isabelle Houbracken, Yves Heremans, Donatienne Tyteca, Ilse Rooman, Luc Bouwens, Frédéric Lemaigre, Patrick Jacquemin. Inflammation enables pancreatic acinar cells to overcome resistance to oncogenic Kras by increasing its expression and plasma membrane localization [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A07.

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 µg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cells RNA. We thus validated the DIE (Digestion, Isolation and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells in vivo and to extract high quality RNA from these cells.

Macrophages play a central role in immune and tissue responses of granulomatous lung diseases induced by pathogens and foreign bodies. Circulating monocytes are generally viewed as central precursors of these tissue effector macrophages. Here, we provide evidence that granulomas derive from alveolar macrophages serving as a local reservoir for the expansion of activated phagocytic macrophages. By exploring lung granulomatous responses to silica particles in IL-1-deficient mice, we found that the absence of IL-1α, but not IL-1β, was associated with reduced CD11b(high) phagocytic macrophage accumulation and fewer granulomas. This defect was associated with impaired alveolar clearance and resulted in the development of pulmonary alveolar proteinosis (PAP). Reconstitution of IL-1α(-/-) mice with recombinant IL-1α restored lung clearance functions and the pulmonary accumulation of CD11b(high) phagocytic macrophages. Mechanistically, IL-1α induced the proliferation of CD11b(low) alveolar macrophages and differentiated these cells into CD11b(high) macrophages which perform critical phagocytic functions and organize granuloma. We newly discovered here that IL-1α triggers lung responses requiring macrophage proliferation and maturation from tissue-resident macrophages. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Hemp (Cannabis sativa) has a highly variable sexual phenotype. In dioecious hemp, the sex is controlled by heteromorphic sex chromosomes according to an X-to-autosomes equilibrium. However, in monoecious hemp, the sex determinism remains widely unknown and has never been related to a quantitative approach of sex expression. The present paper aims to contribute to the comprehension of the sex determinism in monoecious hemp by assessing the genotypic variability of its sex expression and establishing its sex chromosomes. Five monoecious and one dioecious cultivars were grown in controlled conditions under several photoperiods. The monoecy degree of 194 monoecious plants was recorded at each node by a figure ranging from 0 (male flowers only) to 6 (female flowers only). The genome size of 55 plants was determined by flow cytometry. The DNA of 115 monoecious plants was screened with the male-associated marker MADC2. The monoecy degree varied significantly among monoecious cultivars from 3.36 ± 2.28 in 'Uso 31' to 5.70 ± 0.81 in the most feminised 'Epsilon 68'. The variation of monoecy degree among cultivars remained consistent across trials despite a significant "cultivar × trial" interaction and partly agreed with their earliness. The genome size of monoecious plants (1.791 ± 0.017 pg) was not different from that of females (1.789 ± 0.019 pg) but significantly lower than that of males (1.835 ± 0.019 pg). MADC2 was absent from all monoecious plants. These results strongly support that cultivars of monoecious hemp have the XX constitution and that their sex expression has a genetic basis. © 2013 Springer Science+Business Media Dordrecht.

Transcriptional networks control the differentiation of the hepatocyte and cholangiocyte lineages from embryonic liver progenitor cells, and their subsequent maturation to the adult phenotype. However, how accurate levels of hepatocyte and cholangiocyte gene expression are determined during differentiation remains poorly understood. Here we identify miR-337-3p as a novel regulator of liver development. MiR-337-3p stimulates expression of cholangiocyte genes and represses hepatocyte genes in undifferentiated progenitor cells in vitro and in embryonic mouse livers. Beyond the stage of lineage segregation, miR-337-3p controls the transcriptional network dynamics of developing hepatocytes, and balances both cholangiocyte populations that constitute the ductal plate. MiR-337-3p requires Notch and Transforming Growth Factor-β signaling, and exerts a biphasic control on the hepatocyte transcription factor Hepatocyte Nuclear Factor (HNF) 4α, by modulating its activation and repression. With help of an experimentally validated mathematical model we show that this biphasic control results from an incoherent feedforward loop between miR-337-3p and HNF4α. Conclusion: Our results identify miR-337-3p as a novel regulator of liver development, and highlight how tight quantitative control of hepatic cell differentiation is exerted via specific gene regulatory network motifs. This article is protected by copyright. All rights reserved.

Different studies on AGuIX nanoparticles have been achieved in the biomedical domain, showing that they allow us to combine multimodal and theranostic properties in oncology. The targeting of apoptotic cells presents a wide range of biomedical applications, including the monitoring of antitumoral therapy and the diagnosis of diseases related to this process, such as atherosclerosis, ischemia, chronic inflammation or autoimmune disorders. AGuIX nanoparticles functionalized with a peptide that recognizes apoptotic cells and with organic fluorophores were characterized by several physicochemical and biological methods such as HPLC, relaxometry and photon correlation spectroscopy, which attested to their potential as bimodal tracers detected by optical imaging and MRI. An increase of relaxivity and stability of AGuIX nanoparticles is also observed after their vectorization. The biological efficiency of this novel bimodal probe to target apoptotic cells was evaluated by fluorescence microscopy, relaxometry, MRI and flow cytometry on a lymphoblastic human T-cell line. In vitro cell apoptosis was chemically induced by incubation with camptothecin. Our in vitro experiments showed a significant specificity of vectorized AGuIX nanoparticles for camptothecin-treated cells that suggests their potential efficiency as probes to target apoptosis. Copyright © 2016 John WileySons, Ltd.

Macrophages play a central role in immune and tissue responses of granulomatous lung diseases induced by pathogens and foreign bodies. Circulating monocytes are generally viewed as central precursors of these tissue effector macrophages. Here, we provide evidence that granulomas derive from alveolar macrophages serving as a local reservoir for the expansion of activated phagocytic macrophages. By exploring lung granulomatous responses to silica particles in IL-1-deficient mice, we found that the absence of IL-1α, but not IL-1β, was associated with reduced CD11b(high) phagocytic macrophage accumulation and fewer granulomas. This defect was associated with impaired alveolar clearance and resulted in the development of pulmonary alveolar proteinosis (PAP). Reconstitution of IL-1α(-/-) mice with recombinant IL-1α restored lung clearance functions and the pulmonary accumulation of CD11b(high) phagocytic macrophages. Mechanistically, IL-1α induced the proliferation of CD11b(low) alveolar macrophages and differentiated these cells into CD11b(high) macrophages which perform critical phagocytic functions and organize granuloma. We newly discovered here that IL-1α triggers lung responses requiring macrophage proliferation and maturation from tissue-resident macrophages.

Background & Aims Diseases of the exocrine pancreas are often associated with perturbed differentiation of acinar cells. MicroRNAs (miRNAs) regulate pancreas development, yet little is known about their contribution to acinar cell differentiation. We aimed to identify miRNAs that promote and control the maintenance of acinar differentiation. Methods We studied mice with pancreas- or acinar-specific inactivation of Dicer ( Foxa3-Cre/Dicer loxP/− mice), combined (or not) with inactivation of hepatocyte nuclear factor (HNF) 6 ( Foxa3-Cre/Dicer loxP/− /Hnf6−/− mice). The role of specific miRNAs in acinar differentiation was investigated by transfecting cultured cells with miRNA mimics or inhibitors. Pancreatitis-induced metaplasia was investigated in mice after administration of cerulein. Results Inhibition of miRNA synthesis in acini by inactivation of Dicer and pancreatitis-induced metaplasia were associated with repression of acinar differentiation and with induction of HNF6 and hepatic genes. The phenotype of Dicer-deficient acini depends on the induction of HNF6; overexpression of this factor in developing acinar cells is sufficient to repress acinar differentiation and to induce hepatic genes. Let-7b and miR-495 repress HNF6 and are expressed in developing acini. Their expression is inhibited in Dicer-deficient acini, as well as in pancreatitis-induced metaplasia. In addition, inhibiting let-7b and miR-495 in acinar cells results in similar effects to those found in Dicer-deficient acini and metaplastic cells, namely induction of HNF6 and hepatic genes and repression of acinar differentiation. Conclusions Let-7b, miR-495, and their targets constitute a gene network that is required to establish and maintain pancreatic acinar cell differentiation. Additional studies of this network will increase our understanding of pancreatic diseases.

Arsenic trioxide (As2O3) is an effective therapeutic against acute promyelocytic leukemia and certain solid tumors. Because As2O3 inhibits mitochondrial respiration in leukemia cells, we hypothesized that As2O3 might enhance the radiosensitivity of solid tumors by increasing tumor oxygenation [partial pressure of oxygen (pO2)] via a decrease in oxygen consumption. Two murine models of radioresistant hypoxic cancer were used to study the effects of As2O3. We measured pO2 and the oxygen consumption rate in vivo by electron paramagnetic resonance oximetry and 19fluorine-MRI relaxometry. Tumor perfusion was assessed by Patent blue staining. In both models, As2O3 inhibited mitochondrial respiration, leading to a rapid increase in pO2. The decrease in oxygen consumption could be explained by an observed decrease in glutathione in As2O3-treated cells, as this could increase intracellular reactive oxygen species that can disrupt mitochondrial membrane potential. When tumors were irradiated during periods of As2O3-induced augmented oxygenation, radiosensitivity increased by 2.2-fold compared with control mice. Notably, this effect was abolished when temporarily clamped tumors were irradiated. Together, our findings show that As2O3 acutely increases oxygen consumption and radiosensitizes tumors, providing a new rationale for clinical investigations of As2O3 in irradiation protocols to treat solid tumors. Cancer Res; 72(2); 482–90. ©2011 AACR.

The antitumoral properties of endocannabinoids received a particular attention these last few years. Indeed, these endogenous molecules have been reported to exert cytostatic, apoptotic and antiangiogenic effects in different tumor cell lines and tumor xenografts. Therefore, we investigated the cytotoxicity of three N-acylethanolamines – N-arachidonoylethanolamine (anandamide, AEA), N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA) - which were all able to time- and dose-dependently reduce the viability of murine N1E-115 neuroblastoma cells. Moreover, several inhibitors of FAAH and NAAA, whose presence was confirmed by RT-PCR in the cell line, induced cell cytotoxicity and favored the decrease in cell viability caused by N-acylethanolamines. The most cytotoxic treatment was achieved by the co-incubation of AEA with the selective FAAH inhibitor URB597, which drastically reduced cell viability partly by inhibiting AEA hydrolysis and consequently increasing AEA levels. This combination of molecules synergistically decreased cell proliferation without inducing cell apoptosis or necrosis. We found that these effects are independent of cannabinoid, TRPV1, PPARα, PPARγ or GPR55 receptors activation but seem to occur through a lipid raft-dependent mechanism. These findings further highlight the interest of targeting the endocannabinoid system to treat cancer. More particularly, this emphasizes the great potential benefit of designing novel anti-cancerous therapies based on the association of endocannabinoids and inhibitors of their hydrolysis.

Lenalidomide, a daughter molecule of Thalidomide, and IMIDs® are immunomodulatory drugs that have been described as having immunomodulatory properties and anti-tumor activity. The effect of Lenalidomide towards Peripheral Blood Mononuclear Cells (PBMC) has been studied and direct effects towards T cells have been described, such as an increase of interferon-γ (IFN-γ) and interleukin (IL)-2 production. As a consequence, it has been also described that IL-2 subsequently activates Natural Killer (NK) cells. Nevertheless, direct effects of Lenalidomide on NK cells from healthy volunteers have never been described, if searched. Here we show that Lenalidomide can inhibit the production of IFN-γ by NK cells from healthy donors. It also modifies the phenotype of NK cells through a decrease of the expression of Killer cell Immunoglobulin-like Receptors (KIRs) and NKp46. However, we did not detect consequence of these phenotype modifications on the cytotoxic potential of NK cells.

The development of therapeutic monoclonal antibodies (mAbs) has revolutionized the treatment of cancer along the last ten years. The best examples of their therapeutic efficacies have been obtained with rituximab for the treatment of CD20+ B-cell Non-Hodgkin Lymphoma (B-NHL), and several others antibodies with optimized bioactivities are now being developed for the treatment of various malignant hemopathies. We review here the main drugs developed in this field, and present some emerging concepts able to improve the bioactivities of the next generation of therapeutic mAbs.

AHR (Recepteur aux aryls hydrocarbures) est un facteur de transcription qui controle la production d’IL-22 par differents types cellulaires. Precedemment, nous avons mis en evidence le role deletere de l’IL-22 dans un modele de psoriasis induit par l’application d’imiquimod. Dans cette nouvelle etude, nous avons analyse le role de AHR dans ce modele de psoriasis et sur la production d’IL-22 par les cellules de la peau. Nous montrons que des souris deficientes en AHR sont partiellement protegees contre l’acanthose induite par l’application d’imiquimod au niveau des oreilles et que cette protection est associee a une diminution de l’expression d’IL-22 au niveau de la peau. Nous observons cependant que AHR n’est pas absolument requis pour induire l’expression de l’IL-22. En effet, nous montrons que, au niveau de la peau exposee a l’imiquimod, certains lymphocytes T tels que les lymphocytes T γδ ainsi qu’une population de cellules de l’immunite innee sont toujours capables de produire l’IL-22 en absence de AHR. Si la capacite de ces cellules a produire de l’IL-22 n’est pas affectee par l’absence de AHR, c’est leur recrutement dans la peau enflammee qui est significativement diminue. En conclusion, en absence de AHR, une protection partielle contre le developpement d’acanthose en reponse a l’imiquimod est associee a une diminution de l’expression de l’IL-22 qui, elle-meme, est secondaire a un defaut de recrutement des cellules productrices de cette cytokine dans la peau.