TREM1 regulates antifungal immune responses in invasive pulmonary aspergillosis.

Pattern recognition receptors (PRRs) are responsible for Aspergillus fumigatus recognition by innate immunity and its subsequent immune signaling. The triggering receptor expressed on myeloid cells 1 (TREM1) is a recently characterized pro-inflammatory receptor constitutively expressed on the surface of neutrophils and macrophages. A soluble form (sTREM1) of this protein that can be detected in human body fluids has been identified. Here we investigated the role of TREM1 during invasive pulmonary aspergillosis (IPA). IPA patients displayed significantly higher levels of sTREM1 in bronchoalveolar lavages when compared to control patients. Functional analysis in TREM1 showed that the levels of sTREM1 and TREM1 pathway-related cytokines were influenced by single nucleotide polymorphisms in TREM1. In addition, we confirmed a role of TREM1 on antifungal host defense against A. fumigatus in a murine model of IPA. TREM1 deficiency increased susceptibility to infection in the immunosuppressed murine host. Deletion of TREM1 showed delayed innate and adaptive immune responses and impaired pro-inflammatory cytokine responses. The absence of TREM1 in primary macrophages attenuated the TLR signaling by altering the expression of both receptor and effector proteins that are critical to the response against A. fumigatus. In this study, and for the first time, we demonstrate the key role for the TREM1 receptor pathway during IPA.

Notch1 regulates progenitor cell proliferation and differentiation during mouse yolk sac hematopoiesis.

Loss-of-function studies have demonstrated the essential role of Notch in definitive embryonic mouse hematopoiesis. We report here the consequences of Notch gain-of-function in mouse embryo hematopoiesis, achieved by constitutive expression of Notch1 intracellular domain (N1ICD) in angiopoietin receptor tyrosine kinase receptor-2 (Tie2)-derived enhanced green fluorescence protein (EGFP(+)) hematovascular progenitors. At E9.5, N1ICD expression led to the absence of the dorsal aorta hematopoietic clusters and of definitive hematopoiesis. The EGFP(+) transient multipotent progenitors, purified from E9.5 to 10.5 Tie2-Cre;N1ICD yolk sac (YS) cells, had strongly reduced hematopoietic potential, whereas they had increased numbers of hemogenic endothelial cells. Late erythroid cell differentiation stages and mature myeloid cells (Gr1(+), MPO(+)) were also strongly decreased. In contrast, EGFP(+) erythro-myeloid progenitors, immature and intermediate differentiation stages of YS erythroid and myeloid cell lineages, were expanded. Tie2-Cre;N1ICD YS had reduced numbers of CD41(++) megakaryocytes, and these produced reduced below-normal numbers of immature colonies in vitro and their terminal differentiation was blocked. Cells from Tie2-Cre;N1ICD YS had a higher proliferation rate and lower apoptosis than wild-type (WT) YS cells. Quantitative gene expression analysis of FACS-purified EGFP(+) YS progenitors revealed upregulation of Notch1-related genes and alterations in genes involved in hematopoietic differentiation. These results represent the first in vivo evidence of a role for Notch signaling in YS transient definitive hematopoiesis. Our results show that constitutive Notch1 activation in Tie2(+) cells hampers YS hematopoiesis of E9.5 embryos and demonstrate that Notch signaling regulates this process by balancing the proliferation and differentiation dynamics of lineage-restricted intermediate progenitors.

Phospholipid metabolism regulated by a transcription factor sensing phosphatidic acid.

Cells regulate the biophysical properties of their membranes by coordinated synthesis of different classes of lipids. Here, we identified a highly dynamic feedback mechanism by which the budding yeast Saccharomyces cerevisiae can regulate phospholipid biosynthesis. Phosphatidic acid on the endoplasmic reticulum directly bound to the soluble transcriptional repressor Opi1p to maintain it as inactive outside the nucleus. After the addition of the lipid precursor inositol, this phosphatidic acid was rapidly consumed, releasing Opi1p from the endoplasmic reticulum and allowing its nuclear translocation and repression of target genes. Thus, phosphatidic acid appears to be both an essential ubiquitous metabolic intermediate and a signaling lipid.

Selección de los linfocitos T por señales de baja afinidad: el modelo de los superantígenos endógenos de ratón / T lymphocyte selection by low-affinity signals: the model of mouse endogenous superantigens

Durante toda su vida, las células T responden a señales específicas según su afinidad por el antígeno. La mayoría de los estudios sobre las funciones de los linfocitos T han empleado modelos de alta afinidad, que inducen respuestas dramáticas pero que pueden no ser representativos de los procesos fisiológicos. Para comprender estos últimos se necesitan modelos adecuados de baja afinidad. En estos momentos hay disponibles pocos modelos de baja afinidad; uno de ellos es la respuesta a superantígenos endógenos en cepas de ratón "no eliminadoras", que no expresan moléculas IE de clase II. En el ratón, estos superantígenos están codificados por secuencias provirales de "Mouse Mammary Tumour Virus" (MMTV) insertadas en el genoma. Cuando estos superantígenos son presentados por moléculas IA de clase II, inducen una respuesta subóptima de las células T. El modelo de respuesta de baja afinidad a superantígenos endógenos evita los problemas ligados a la utilización de ratones transgénicos para el TCR, y ya ha permitido identificar distintos factores que controlan las respuestas de baja afinidad y que no afectan a las de alta afinidad. Especialmente, y entre otros resultados, han permitido detectar una regulación específica de las células T + por las B y T +. Los resultados obtenidos podrían ser aplicables a otras interacciones de baja afinidad, que son de especial interés en el desarrollo de procesos de autoinmunidad (AU)

Effect of phenanthrene and Rhodotorula glutinis on arbuscular mycorrhizal fungus colonization of maize roots.

The effect of the polycyclic aromatic hydrocarbon (PAH) phenanthrene and the yeast Rhodotorula glutinis on the arbuscular mycorrhizal fungus (AMF) Glomus geosporum colonizing maize roots, was studied. During a 90-day experiment, the highest G. geosporum colonization values were found in control plants. Mycorrhiza root length, measured both on the basis of percentage of root colonization and on the activity of succinate dehydrogenase, showed similar patterns in different phenanthrene treatments. The presence of phenanthrene in the substrate reduced G. geosporum intraradical colonization. The presence of R. glutinis did not enhance AMF colonization in the presence of phenanthrene. The biomass of the external mycelium estimated on the basis of the fatty acid 16:1 omega 5 concentration showed a progressive increase through time, and the amounts of this fatty acid differed among treated and untreated substrates. However, this increase was found to be lowest in the phenanthrene and Rhodotorula treatment at 60 days. There was less phenanthrene accumulation in roots of maize inoculated with AMF and the yeast than in roots inoculated only with AMF. A similar pattern was observed in the phenanthrene content of G. geosporum spores collected after 90 days.

Long-lived polyclonal B-cell lines derived from midgestation mouse embryo lymphohematopoietic progenitors reconstitute adult immunodeficient mice.

Lymphohematopoietic progenitors derived from midgestation mouse embryos were established in long-term cultures with stromal cell monolayers and interleukin 7 (IL-7), giving rise to B-lineage cell lines. The initial emergence and in vitro establishment of these early embryo cell lines were highly sensitive to IL-7-mediated signals, in comparison to cell lines similarly obtained using precursors from late fetal liver (> 13 days postcoitum) and adult bone marrow. The early embryo-derived progenitors spontaneously differentiated in vitro to CD19(+)IgM(+) immature B cells in the presence of optimal concentrations of IL-7, in contrast to those progenitors obtained from late gestation and adult mice, whose differentiation only occurred in the absence of IL-7. The newly in vitro-generated B cells of the early embryo cell lines repopulated adult immunodeficient severe combined immunodeficient mice on their adoptive transfer in vivo and generated specific humoral immune responses after immunization.

Both B and gammadelta TCR(+) lymphocytes regulate alphabeta TCR(+) lymphocytes involved in superantigen specific responses.

Endogenous superantigens (SAg) presented by MHC class II IA molecules induce slow-evolving negative selection of alpha beta T cells. The role of both B and gamma delta T cells on the regulation of these SAg-specific alpha beta T cell responses was addressed in IA(b+)IE(b-) C57BL/6 mice bearing genetically induced B cell and gamma delta T cell deficiencies. B lymphocytes were required in the negative selection of Vbeta5(+)/Vbeta12(+) CD4(+) T cells. In contrast, gamma delta T cells positively stimulated the utilization of the same SAg-responsive alpha beta T cell subsets. These differences started in mature CD4(+) thymocytes and extended to naive T cell pools for B cell negative selection, and up to memory T cells for gamma deltaT cell influences. The levels of SAg-responsive T cells did not vary between C57BL/6 and double deficient (B cell and gamma delta T cell-deficient) congenic mice, implying that both B and gamma delta T cells acted through independent mechanisms.

Fluorescein diacetate hydrolysis as a measure of fungal biomass in soil.

The fatty acid methyl esters of lipids extracted from an agricultural soil in the preharvest period of soybean or middle growth cycle from wheat were characterized and quantified by gas-liquid chromatography. The fatty acids 18:2omega6 and 16:1omega5 were used as markers of saprotrophic and arbuscular mycorrhizal fungi. In parallel, biomass estimation through plate counts in selective media for cellulolytic and saprotrophic fungi was also performed all throughout a soybean crop or middle growth cycle of wheat. As an enzymatic method, the fluorescein diacetate (FDA) hydrolytic activity of the samples was determined. Owing to the high relationship exhibited by FDA hydrolysis with organic carbon and total nitrogen content of soil, the enzymatic activity was correlated with the microbial biomass estimated through marker lipids or plate counts. The results obtained point out that FDA hydrolysis may be used as a rapid, cheap, and reliable estimator of fungal biomass.

Influence of prolactin on the differentiation of mouse B-lymphoid precursors.

Development and activation of immune cells are submitted to hormonal influences, as illustrated by the roles of corticosteroids in thymus, pregnancy-related estrogens in B-cell development, or prolactin (PRL) on T-cell generation and function. We have analyzed the putative role of PRL in B lymphopoiesis and differentiation. We chose as an experimental model the interleukin (IL)-3 dependent BaF-3 pro-B cell line, which was transfected with the rat long form of the PRL receptor (PRL-R) and transferred from IL-3- to PRL-enriched media. When stimulated with PRL, the PRL-R transfectants underwent some changes characteristic of B-cell differentiation: (a) IL-2R alpha chain became positively controlled by PRL; (b) antiapoptotic Bcl-2 protein was induced by PRL in a dose-dependent manner; and (c) transcription of the pre-B cell receptor encoding the lambda5 gene was strongly up-regulated. We attempted to evaluate the differentiation-promoting activity of PRL in more physiological conditions, and the presence of PRL-R in bone marrow B-cell precursors was revealed. Furthermore, PRL promoted significant expansions of defined B-lineage cell populations in short-term bone marrow cell cultures. These findings suggest that PRL, in collaboration with other cytokines and hormonal influences, modulates B-cell development.

Differential involvement of the transcription factor Blimp-1 in T cell-independent and -dependent B cell differentiation to plasma cells.

Along humoral immune responses, different stimuli drive the differentiation of B lymphocytes to Ig-secreting plasma cells in discrete microenvironments. The Blimp-1 transcription factor is up-regulated early during the transition of mature B cells to IgM-secreting plasma cells. In the present study, we have examined the requirement of Blimp-1 in plasma cell formation after both T cell-independent (LPS) and -dependent (CD40 + IL-4, Th cell lines) stimulation of spleen B cells. B lymphocyte-induced maturation protein (Blimp-1) was expressed early after in vitro LPS stimulation, mainly in a population of IgM+Syndecan+CD43+ preplasma cells. In contrast, the BSAP transcription factor expressed in mature B cells was down-regulated during the differentiation to plasma cells. Treatment of these cultures with Blimp-1-specific antisense phosphorothioate oligonucleotides suppressed both Blimp-1 protein levels and the emergence of IgM+Syndecan+ cells and plasma cells. However, T-B cell cocultures of spleen B cells from C3H/HeJ (H-2k) mice and syngeneic autoreactive SR.10 Th2 cells submitted to the anti-Blimp-1 therapy did not show any significant reduction in IgM- and IgG1-secreting plasma cell formation. Spleen B cells treated with anti-CD40 mAb + IL-4 differentiated to IgG1-secreting cells without significant transcription of the Blimp-1 gene; anti-Blimp-1 treatment subsequently did not have any effect in the later cultures. Altogether, these results suggest that Blimp-1 transcription factor specifically promotes T cell-independent B cell differentiation to plasma cells, probably at preplasma cell stages. In contrast, T cell-dependent plasma cell formation likely evolves through Blimp-1-independent pathways.

Developmental hematopoiesis.

Hematopoiesis is a developmental process that evolves throughout the lifespan of an individual. Most work in the field has focused on events occurring in the adult bone marrow (BM). In the embryo, blood and endothelial cell generation begins very early after gastrulation, in defined intraembryonic mesodermic sites. Recent multidisciplinary studies, taking advantage of classic embryological and gene targeting technology in various species, have provided a new image of embryofetal lymphohemopoiesis, which includes the suggestion of developmental compartmentalization or waves. The first hematopoietic stem cells (HSC) migrate further and home in an ordered sequence of supporting microenvironments depending on scarcely known molecular requirements. These early hematopoietic progenitors show important differences in their cell biology and differentiation potentialities with respect to those present in adult stages; this fact, together with specific microenvironmental influences, define a process that diverges significantly from that occurring in the BM. Here, we update the latest developments in the field, the new understanding of lymphohemopoiesis in prenatal life, and the novel questions that this emerging paradigm is producing.

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase.

p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

Antigenic phenotype and gene expression pattern of lymphohemopoietic progenitors during early mouse ontogeny.

Hemopoiesis, initiated in the early embryo yolk sac (YS) (7.5-8 days postcoitum (pc) in mouse), takes place thereafter in sites successively seeded by extrinsic hemopoietic stem cells (HSC). Since the existence of intraembryonic HSC has been proven experimentally in some vertebrates, it is also likely that not all HSC originate in the YS in mammals, as previously thought. Candidate intraembryonic sites that may be active in producing HSC before liver colonization are the para-aortic splanchnopleura (P-Sp) and the aorta-gonads-mesonephros region (AGM). Here we explore these sites directly for the presence of cells with hemopoietic-specific surface molecules and gene activities. The Ags c-kit, AA4.1, Mac-1, and Sca-1 begin to be expressed on some P-Sp and AGM cells, making it possible to distinguish subpopulations that evolve according to reproducible developmental patterns. On the basis of RAG-1 gene transcription, the first lymphoid precursors in the mouse embryo appear to be present 9.5 to 10 days pc in P-Sp/AGM and YS. Starting B-cell lymphopoiesis (9-12 days pc) is characterized by nonexpression of the surrogate light chain lambda 5-encoding gene and biased usage of IgH DJ4 rearrangements. In the 12.5- to 13.5-day-pc fetal liver (FL), a switch occurs, characterized by the random use of all IgH DJ and the detection of lambda 5 gene transcripts.

Isolation of peritoneal precursors of B-1 cells in the adult mouse.

Two weeks of daily peritoneopheresis of adult mice result in the selective depletion of B-1 cells, followed by the appearance of a population of B220+IgM-lymphocytes in the peritoneal cavity. These cells share with bone marrow (BM) pre-B cells expression of lambda 5, VpreB, and RAG-1 genes and a higher fraction of unrearranged V to DJ heavy (H) chain immunoglobulin (Ig) gene segments, when compared with mature B lymphocytes. Upon transfer to SCID recipients, sorted peritoneal B220+IgM- cells fail to colonize the BM, repopulate very few B cells in the spleen, but entirely reconstitute the B-1 cell compartment in the peritoneal and pleuropericardial cavities. In contrast, parallel transfers of sorted BM and pleuropericardial cavities. In contrast, parallel transfers of sorted BM B220+IgM- cells result in reconstitution of the BM and spleen B lineage cell compartments, but in no coelomic B cell repopulation. Both types of pre-B cells reconstitute splenic plasma cells of donor origin, but with markedly distinct efficiencies: the ratio of IgM-plasma cell/B cell numbers in the spleens of peritoneal pre-B cell recipients is more than 500-fold higher than that of recipients reconstituted by BM pre-B cells. We take these data to indicate that (1) differentiative commitment to the B-1 cell population occurs before selection events on mature cells; (2) B-1 precursors exist or may be locally produced in the adult mouse; (3) there is a lineage-related differential ability of mature B cells to undergo terminal differentiation to high-rate Ig secretion.

A single base deletion in the Tfm androgen receptor gene creates a short-lived messenger RNA that directs internal translation initiation.

Testosterone-resistant male mice hemizygous for the X-chromosome-linked mutant gene Tfm express detectable but severely reduced levels of androgen receptor mRNA, amounting to about 10% of the level found in normal male littermates. No structural abnormality could be identified in the coding region of the messenger by a series of RNase-protection assays. However, cell-free translation of RNAs transcribed in vitro from enzymatically amplified overlapping segments of exon 1 revealed a truncated receptor protein and helped to localize the site of premature termination. Sequence analysis of the relevant DNA segment disclosed that deletion of a single nucleotide in the hexacytidine stretch at position 1107-1112 alters the reading frame of the messenger and introduces 41 missense amino acids before a premature termination codon at position 1235-1237. Separately initiated carboxyl-terminal polypeptides are synthesized in vitro, starting probably at the in-frame AUG codon 1507-1509, which lies in a favorable context for translation initiation, and at the non-AUG codon 1144-1146. Transcriptional impairments of the Tfm gene were ruled out by a quantitative analysis of enzymatically amplified nuclear RNA precursors. No other change could be identified by sequencing the complete coding region of Tfm cDNA. The finding of the unsuspected termination codon and the evidence of internally initiated carboxyl-terminal polypeptides reconcile previous conclusions and account for all known phenotypic properties of the mutation.

Selective expansion of idiotype sharing T and B cells in cyclosporin A-mediated autoimmunity.

CBA/N mice submitted to autologous bone marrow reconstitution after lethal irradiation and simultaneous Cyclosporin A (CsA) treatment develop a chronic graft-versus-host disease with autoimmune characteristics. When compared to normal controls, diseased mice show an overrepresentation of V beta 8-expressing T cells (65-80% of all CD3+ lymphocytes), together with a marked increase in the titres of serum Ig that specifically bind to F(ab')2 fragments of anti-V beta 8 F23.1 antibodies. Such 'V beta 8-like' Ig V regions are abundantly represented among the IgG2b and mAbs of an unselected collection of hybridomas derived from these mice. These mAbs are not multireactive Ig as they fail to bind to a panel of various antigens and antibodies, but often show simultaneous reactivity with anti-idiotypic mAbs to F23.1 and auto-binding. These molecules may provide the structural basis of V-region specific complementarities, driving the expansion of restricted T and B cell repertoires associated with pathological autoimmunity.

[Structure of messenger RNA of androgen receptor in mice and molecular characterization in Tfm mutant]. / Structure de l'ARN messager du récepteur des androgènes de Souris et caractérisation moléculaire du mutant Tfm.

Complementary DNA clones covering the complete coding region of the mouse androgen receptor were assembled by enzymatic amplification (PCR) from testicular RNA and genomic DNA and fully sequenced. The deduced amino acid sequence departs from the rat sequence at 21 positions, 20 of which are in the amino-terminal trans-activation domain. A single 10 kb long messenger RNA containing a 3' noncoding portion longer than 5 kb was detected. The murine cDNA sequence provided the basis to examine the testicular feminization (Tfm) mutation at the molecular level. The androgen receptor messenger RNA level was found reduced about 10-fold in the Tfm mice. The expression of the mutant receptor is affected at a post-transcriptional level. A single base deletion in the hexanucleotide stretch 1107-1112, not far from the 3' end of exon 1, introduces a frame-shift that leads to premature termination of the AR protein. Separately initiated polypeptides containing the DNA-binding and the steroid-binding domains of the androgen receptor are produced in vitro by using strong internal translation initiation sites. These carboxyl-terminal polypeptides have the characteristics of the shortened form of the receptor previously described in the tissues of Tfm mice.

Structure and size distribution of the androgen receptor mRNA in wild-type and Tfm/Y mutant mice.

Complementary DNA clones covering the coding region of the mouse androgen receptor (AR) were assembled by enzymatic amplification from testicular RNA and genomic DNA. The deduced amino acid sequence consists of 899 residues and departs from the rat sequence at 21 positions, 20 of which are in the amino-terminal trans-activation domain. A notable cluster of substitutions lies in the region of the long glutamine repeat at positions 174-195. The size heterogeneity of AR messengers suggested by previous blot hybridization experiments was examined by RNase protection analysis of sucrose gradient-fractionated poly(A) RNA from mouse liver. A predominant 10-kilobase long mRNA species was found to encode the AR, and a 3' noncoding portion longer than 5 kilobases was demonstrated by internal cleavage with RNase-H, followed by blot hybridization with a 3' probe. The sensitivity afforded by the use of homologous RNA probes in solution hybridizations allowed the demonstration in Tfm/Y mutant mice of an AR mRNA that covers the entire coding region, but is present at 10- to 20-fold lower levels than in normal animals. The detection of significant amounts of receptor messenger revives earlier suggestions of an AR protein in Tfm/Y mice and indicates, at variance with other conclusions, that the expression of this mutant AR is affected at a post-transcriptional level.