Epithelial cells maintain memory of prior infection with Streptococcus pneumoniae through di-methylation of histone H3.

Epithelial cells are the first point of contact for bacteria entering the respiratory tract. Streptococcus pneumoniae is an obligate human pathobiont of the nasal mucosa, carried asymptomatically but also the cause of severe pneumoniae. The role of the epithelium in maintaining homeostatic interactions or mounting an inflammatory response to invasive S. pneumoniae is currently poorly understood. However, studies have shown that chromatin modifications, at the histone level, induced by bacterial pathogens interfere with the host transcriptional program and promote infection. Here, we uncover a histone modification induced by S. pneumoniae infection maintained for at least 9 days upon clearance of bacteria with antibiotics. Di-methylation of histone H3 on lysine 4 (H3K4me2) is induced in an active manner by bacterial attachment to host cells. We show that infection establishes a unique epigenetic program affecting the transcriptional response of epithelial cells, rendering them more permissive upon secondary infection. Our results establish H3K4me2 as a unique modification induced by infection, distinct from H3K4me3 or me1, which localizes to enhancer regions genome-wide. Therefore, this study reveals evidence that bacterial infection leaves a memory in epithelial cells after bacterial clearance, in an epigenomic mark, thereby altering cellular responses to subsequent infections and promoting infection.

Streptococcus pneumoniae drives specific and lasting Natural Killer cell memory.

NK cells are important mediators of innate immunity and play an essential role for host protection against infection, although their responses to bacteria are poorly understood. Recently NK cells were shown to display memory properties, as characterized by an epigenetic signature leading to a stronger secondary response. Although NK cell memory could be a promising mechanism to fight against infection, it has not been described upon bacterial infection. Using a mouse model, we reveal that NK cells develop specific and long-term memory following sub-lethal infection with the extracellular pathogen Streptococcus pneumoniae. Memory NK cells display intrinsic sensing and response to bacteria in vitro, in a manner that is enhanced post-bacterial infection. In addition, their transfer into naïve mice confers protection from lethal infection for at least 12 weeks. Interestingly, NK cells display enhanced cytotoxic molecule production upon secondary stimulation and their protective role is dependent on Perforin and independent of IFNγ. Thus, our study identifies a new role for NK cells during bacterial infection, opening the possibility to harness innate immune memory for therapeutic purposes.

Kidney functional morphology variations between spring and winter in the Saharan male lizard Uromastyx acanthinura (Sauria, Agamidae), with special reference to body water economy.

To better understand the adaptive mechanisms in Uromastyx acanthinura to the seasonal variations in the arid environment, the present study aimed to explore the kidney functional morphology involved in body water economy. These investigations were carried out by the histological, histochemical and immuno-histochemical methods using conventional light microscopy. The glomeruli number is estimated at 2000 per kidney. The glomeruli size is rather small and decreases significantly in winter. Interestingly, the proximal convoluted tubule (PCT) is long and divided into two different segments which is one of the particularities of this species. Both of the distal convoluted tubule (DCT), connecting tubule (CnT) and collecting duct (CD) epithelium contains mucous cells. The nature and intensity of these mucous secretions vary according to seasons. The evident hypertrophy of the secondary collecting duct (SCD) and tertiary collecting duct (TCD) epithelium is related to the high secretory activity during spring, corresponding to the sexual segment of kidney (SSK). Labeling with anti α-smooth muscle actin-1 showed a thick layer of mucularis surrounded the entire CD. Also, the mesangium of glomeruli contains myofibroblasts. All these renal structural characteristics involved in body water economy may be considered as an adaptive mechanisms of U. acanthinura to resist to dehydration and cope with seasonal variations in the arid environment.

Streptococcus pneumoniae Infection Promotes Histone H3 Dephosphorylation by Modulating Host PP1 Phosphatase.

Pathogenic bacteria can alter host gene expression through post-translational modifications of histones. We show that a natural colonizer, Streptococcus pneumoniae, induces specific histone modifications, including robust dephosphorylation of histone H3 on serine 10 (H3S10), during infection of respiratory epithelial cells. The bacterial pore-forming toxin pneumolysin (PLY), along with the pyruvate oxidase SpxB responsible for H2O2 production, play important roles in the induction of this modification. The combined effects of PLY and H2O2 trigger host signaling that culminates in H3S10 dephosphorylation, which is mediated by the host cell phosphatase PP1. Strikingly, S. pneumoniae infection induces dephosphorylation and subsequent activation of PP1 catalytic activity. Colonization of PP1 catalytically deficient cells results in impaired intracellular S. pneumoniae survival and infection. Interestingly, PP1 activation and H3S10 dephosphorylation are not restricted to S. pneumoniae and appear to be general epigenomic mechanisms favoring intracellular survival of pathogenic bacteria.

H3K4me1 Supports Memory-like NK Cells Induced by Systemic Inflammation.

Natural killer (NK) cells are unique players in innate immunity and, as such, an attractive target for immunotherapy. NK cells display immune memory properties in certain models, but the long-term status of NK cells following systemic inflammation is unknown. Here we show that following LPS-induced endotoxemia in mice, NK cells acquire cell-intrinsic memory-like properties, showing increased production of IFNγ upon specific secondary stimulation. The NK cell memory response is detectable for at least 9 weeks and contributes to protection from E. coli infection upon adoptive transfer. Importantly, we reveal a mechanism essential for NK cell memory, whereby an H3K4me1-marked latent enhancer is uncovered at the ifng locus. Chemical inhibition of histone methyltransferase activity erases the enhancer and abolishes NK cell memory. Thus, NK cell memory develops after endotoxemia in a histone methylation-dependent manner, ensuring a heightened response to secondary stimulation.

Epigenetic down-regulation of the HIST1 locus predicts better prognosis in acute myeloid leukemia with NPM1 mutation.

BACKGROUND: The epigenetic machinery is frequently altered in acute myeloid leukemia. Focusing on cytogenetically normal (CN) AML, we previously described an abnormal H3K27me3 enrichment covering 70 kb on the HIST1 cluster (6.p22) in CN-AML patient blasts. Here, we further investigate the molecular, functional, and prognosis significance of this epigenetic alteration named H3K27me3 HIST1 in NPM1-mutated (NPM1mut) CN-AML. RESULTS: We found that three quarter of the NPM1mut CN-AML patients were H3K27me3 HIST1high. H3K27me3 HIST1high group of patients was associated with a favorable outcome independently of known molecular risk factors. In gene expression profiling, the H3K27me3 HIST1high mark was associated with lower expression of the histone genes HIST1H1D, HIST1H2BG, HIST1H2AE, and HIST1H3F and an upregulation of genes involved in myelomonocytic differentiation. Mass spectrometry analyses confirmed that the linker histone protein H1d, but not the other histone H1 subtypes, was downregulated in the H3K27me3 HIST1high group of patients. H1d knockdown primed ATRA-mediated differentiation of OCI-AML3 and U937 AML cell lines, as assessed on CD11b/CD11c markers, morphological and gene expression analyses. CONCLUSIONS: Our data suggest that NPM1mut AML prognosis depends on the epigenetic silencing of the HIST1 cluster and that, among the H3K27me3 silenced histone genes, HIST1H1D plays a role in AML blast differentiation.

Infection Reveals a Modification of SIRT2 Critical for Chromatin Association.

Sirtuin 2 is a nicotinamide-adenine-dinucleotide-dependent deacetylase that regulates cell processes such as carcinogenesis, cell cycle, DNA damage, and infection. Subcellular localization of SIRT2 is crucial for its function but is poorly understood. Infection with the bacterial pathogen Listeria monocytogenes, which relocalizes SIRT2 from the cytoplasm to the chromatin, provides an ideal stimulus for the molecular study of this process. In this report, we provide a map of SIRT2 post-translational modification sites and focus on serine 25 phosphorylation. We show that infection specifically induces dephosphorylation of S25, an event essential for SIRT2 chromatin association. Furthermore, we identify a nuclear complex formed by the phosphatases PPM1A and PPM1B, with SIRT2 essential for controlling H3K18 deacetylation and SIRT2-mediated gene repression during infection and necessary for a productive Listeria infection. This study reveals a molecular mechanism regulating SIRT2 function and localization, paving the way for understanding other SIRT2-regulated cellular processes.

Electrochemical Attachment of Diazonium-Generated Films on Nanoporous Gold.

Nanoporous gold provides a high surface area platform for further chemistry, but the stability of the molecular linkages to the surface will limit applications. We attached aryl molecular layers to nanoporous gold electrodes through electrochemical reduction of the corresponding aryl-diazonium salt and studied the properties and stability of the resulting films in varied attachment conditions. Infrared reflection absorption spectroscopy and X-ray photoelectron spectroscopy were used to confirm the presence of the molecular layers. X-ray photoelectron spectroscopy indicates that the molecular layer is thick and that attachment conditions can form multilayers. However, cyclic voltammetry shows that the multilayers do not block electrochemical activity at the nanoporous gold surface. The molecular layers are resistant to replacement by alkane-thiol chains and exhibit some stability with respect to applied potential. These results indicate that a thick but highly defective molecular film forms with a mixture of strongly and weakly bound molecules.

Expression of fluorescent proteins in Branchiostoma lanceolatum by mRNA injection into unfertilized oocytes.

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Preparation and delivery of 4-hydroxy-tamoxifen for clonal and polyclonal labeling of cells of the surface ectoderm, skin, and hair follicle.

To study the cell behavior during morphogenesis of mouse surface ectoderm, skin, and hair follicles, we (1-3) have developed a new method to temporally induce clones that is based on a tamoxifen-dependent Cre recombinase. The classical protocol consisting in dissolving 4-hydroxy-tamoxifen or tamoxifen in corn oil to perform intraperitoneal (ip) injections (4) is not optimal to control the pharmacokinetic parameters of the induction as it leads to experimental variability in terms of timing and level of induction. We have developed a new protocol that consists in solubilizing 4-OHT or tamoxifen in an aqueous solvent using Cremophor(®) EL (5). This allows for intravenous (iv) and intraperitoneal injections.

Postnatal changes in testicular development and androgen receptors immunolocalization in D'Man ram lambs.

The purpose of this study was to characterize testicular development in D'Man ram lambs, focusing primarily on androgen receptors (ARs) immunolocalization in the adenohypophysis and testis that is not still known in the D'Man ram lamb. Lambs (n = 12) were divided into four groups (three lambs per group). Adenohypophysis and testis were fixed and paraffin embedded; cross-section (3 µm) were stained and evaluated with immunohistochemistry. Testis weight increased at a greater rate between two and five months after birth, which was associated with remarkable changes in testicular histology, including significant increases in the diameter of seminiferous tubules. Spermatogenesis started between three and five months after birth; lumen and elongated spermatids were observed for the first time in three and four months-old animals respectively. ARs detected with immunohistochemistry were located in the nuclei and cytoplasm of adenohypophysis cells, and only in nuclei of testis cells (Leydig, Sertoli, peritubular myoid and germ cells).

Reproductive biology of the endangered percid Zingel asper in captivity: a histological description of the male reproductive cycle.

The endemic Rhodanian percid Zingel asper (Linnaeus, 1758), is usually found throughout the Rhône basin, but this fish is now in sharp decline. Understanding its reproductive physiology is important so as to be able to artificially control its reproduction with a view to re-introducing it. This study was carried out on a population obtained by artificial fertilization and bred in external tanks. Fishes were observed from the juvenile stages through to adulthood. Patterns of testicular development were defined from histological observations. Testes of Z. asper were paired, elongated and fusiform dorsocaudal organs. The two lobes of each gonad joined together to form a duct that extended to the urogenital papillae. They showed a lobular structure. The testicular lobules were of the unrestricted spermatogonial type. Up to 10 months-old, most of the males were immature: their testes showed only type A spermatogonia. The appearance of type B spermatogonia in the lobules of a testis indicated the beginning of spermatogenesis in 10 months-old fish. Spermiogenesis occurred 24 months after the fertilization and, in 26 months-old fish, the cyst opened and released spermatozoa into the lumen of lobules. The spermiation belonged to a cystic type. During the third year, histological observations pointed to the same evolution of adult gonads as during the second year. Sexual maturity was reached in captive Z. asper after two years. The spawning occurred in May in the breeding conditions.