Pharmacological modulation of LMNA SRSF1-dependent splicing abrogates diet-induced obesity in mice.

Bakground/Objectives:Intense drug discovery efforts in the metabolic field highlight the need for novel strategies for the treatment of obesity. Alternative splicing (AS) and/or polyadenylation enable the LMNA gene to express distinct protein isoforms that exert opposing effects on energy metabolism and lifespan. Here we aimed to use the splicing factor SRSF1 that contribute to the production of these different isoforms as a target to uncover new anti-obesity drug. SUBJECTS/METHODS: Small molecules modulating SR protein activity and splicing were tested for their abilities to interact with SRSF1 and to modulate LMNA (AS). Using an LMNA luciferase reporter we selected molecules that were tested in diet-induced obese (DIO) mice. Transcriptomic analyses were performed in the white adipose tissues from untreated and treated DIO mice and mice fed a chow diet. RESULTS: We identified a small molecule that specifically interacted with the RS domain of SRSF1. ABX300 abolished DIO in mice, leading to restoration of adipose tissue homeostasis. In contrast, ABX300 had no effect on mice fed a standard chow diet. A global transcriptomic analysis revealed similar profiles of white adipose tissue from DIO mice treated with ABX300 and from untreated mice fed a chow diet. Mice treated with ABX300 exhibited an increase in O2 consumption and a switch in fuel preference toward lipids. CONCLUSIONS: Targeting SRSF1 with ABX300 compensates for changes in RNA biogenesis induced by fat accumulation and consequently represents a novel unexplored approach for the treatment of obesity.

Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells.

The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1ß and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11-IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

Humanized immune system (HIS) mice as a tool to study human NK cell development.

The study of human hematopoiesis is conditioned by access to nondiseased human tissue samples that harbor the cellular substrates for this developmental process. Technical and ethical concerns limit the availability to tissues derived from the fetal and newborn periods, while adult samples are generally restricted to peripheral blood. Access to a small animal model that faithfully recapitulates the process of human hematopoiesis would provide an important tool. Natural killer (NK) cells comprise between 10% and 15% of human peripheral blood lymphocytes and appear conserved in several species. NK cells are implicated in the recognition of pathogen-infected cells and in the clearance of certain tumor cells. In this chapter, we discuss NK cell developmental pathways and the use of humanized murine models for the study of human hematopoiesis and, in particular, human NK cell development.

Heat shock treatment increases engraftment of transplanted human myoblasts into immunodeficient mice.

Myoblast transfer therapy (MTT) is a strategy that has been proposed to treat some striated muscle pathologies. However, the first therapeutic trials using this technique were unsuccessful due to the limited migration and early cell death of the injected myoblasts. Various strategies have been considered to increase myoblast survival in the host muscle after MTT. Overexpression of heat shock proteins (HSPs) in mouse myoblasts has been shown to improve cell resistance against apoptosis in vitro and in vivo. Our objective was to determine whether heat shock (HS) treatment increased the survival of human myoblasts leading to better participation of the injected cells in muscle regeneration. For this study, HS-treated human myoblasts were injected into the tibialis anterior (TA) muscles of immunodeficient RAG-/- gammaC-/- mice. TA muscles were excised at 24 hour and at 1 month after injection. Our results showed that HS treatment increased the expression of the hsp70 protein and protected the cells from apoptosis in vitro. HS treatment dramatically increased the number of human fibers present at 1 month after injection when compared with nontreated cells. Interestingly, HS treatment decreased apoptosis at 24 hour after human myoblast injection, but no differences were observed concerning proliferation, suggesting that the increased fiber formation among the HS-treated group was probably due to decreased cell death. These data suggested that HS treatment might be used in the clinical context to improve the success of MTT.

Extended amplification in vitro and replicative senescence: key factors implicated in the success of human myoblast transplantation.

The limited success of human myoblast transplantation has been related to immune rejection, poor survival, and limited spread of injected myoblasts after transplantation. An important issue that has received little attention, but is nevertheless of fundamental importance in myoblast transplantation protocols, is the proliferative capacity of human satellite cells. Previous studies from our laboratory have demonstrated that the maximum number of divisions that a population of satellite cells can make decreases with age during the first two decades of life then stabilizes in adulthood. These observations indicate that when satellite cells are used as vectors in myoblast transplantation protocols it is important to consider donor age and the number of divisions that the cells have made prior to transplantation as limiting factors in obtaining an optimal number of donor derived muscle fibers. In this study, myoblasts derived from donors of different ages (newborn, 17 years old, and 71 years old) were isolated and amplified in culture. Their potential to participate in in vivo muscle regeneration in RAG2(-/-)/gamma(c)/C5 triple immunodeficient hosts after implantation was evaluated at 4 and 8 weeks postimplantation. Our results demonstrate that prolonged amplification in culture and the approach to replicative senescence are both important factors that may condition the success of myoblast transplantation protocols.

Non-24-hour sleep-wake syndrome following a car accident.

The authors report the case of a 39-year-old sighted woman who displayed non-24-hour sleep-wake cycles following a car accident. The phase relationship between endogenous circadian markers such as plasma melatonin and 6-sulfatoxymelatonin rhythms and self-selected sleep times was abnormal. A laboratory investigation indicated that she was sensitive to bright light as a circadian synchronizer. MRI and brain CT scans were normal, but microscopic brain damage in the vicinity of the suprachiasmatic nucleus or its output pathways is plausible.

Individualism and collectivism: cultural orientation in locus of control and moral attribution under conditions of social change.

This study examined the validity of the view that the constructs of individualism and collectivism are coherent cultural manifestations necessarily reflected in an individual's attribution patterns. It was hypothesized that the attribution patterns of locus of control and moral accountability would show divergent individualistic and collectivistic influences in a culture during change from a collectivist culture to an individualist culture. 98 university students from the United States and Puerto Rico were administered the Singelis Individualism-Collectivism Scale, Rotter's Locus of Control Scale, and Miller and Luthar's justice-related moral accountability vignettes. Contrary to expectation, the Puerto Rican sample scored less external in locus of control than the United States sample. No cultural differences in moral accountability were found. No strong correlations were found among the variables at the individual level of analysis. Accounting for these results included the lack of representativeness of the samples, the independence of relation between variables at different levels of analysis, and social change.

TCRA gene rearrangement in immature thymocytes in absence of CD3, pre-TCR, and TCR signaling.

During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pTalpha and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, alphabeta(+) thymocytes do develop in pTalpha-deficient mice, showing that TCRalpha-chain genes are rearranged, either in CD4(-)CD8(-) or CD4(+)CD8(+) thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pTalpha and gammac expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and gammac signaling. However, CD3 expression was found to play a role in transcription of rearranged TCRalpha-chain genes in CD4(-)CD8(-) thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.

IL-12-independent IFN-gamma production by T cells in experimental Chagas' disease is mediated by IL-18.

IL-12p35-deficient (IL-12p35(-/-)) mice were highly susceptible to Trypanosoma cruzi infection and succumbed during acute infection, demonstrating the crucial importance of endogenous IL-12 in resistance to experimental Chagas' disease. Delayed immune responses were observed in mutant mice, although comparable IFN-gamma and TNF-alpha blood levels as in wild-type mice were detected 2 wk postinfection. In vivo and in vitro analysis demonstrated that T cells, but not NK cells, were recruited to infected organs. Analysis of mice double deficient in the recombinase-activating gene 2 (RAG2) and IL-12p35, as well as studies involving T cell depletion, identified CD4(+) T cells as the cellular source for IL-12-independent IFN-gamma production. IL-18 was induced in IL-12p35(-/-) mice and was responsible for IFN-gamma production, as demonstrated by in vivo IL-18 neutralization studies. In conclusion, evidence is presented for an IL-12-independent IFN-gamma production in experimental Chagas' disease that is T cell and IL-18 dependent.

Intraembryonic, but not yolk sac hematopoietic precursors, isolated before circulation, provide long-term multilineage reconstitution.

The relative contribution of yolk sac and intraembryonic precursors to hematopoiesis has been a matter of long-standing controversy. As reconstitution activity has so far only been found in embryonic tissues after the onset of circulation, the origin of reconstituting cells could not be formally established. Here, we separated yolk sac and intraembryonic splanchnopleura prior to circulation and maintained the explants in organ culture before transfer. Precursors derived from the intraembryonic site generated multilineage hematopoietic progeny in adult mice for more than 6 months. Yolk sac cells only provided myeloid short-term reconstitution. The results reveal a differential hematopoietic capacity of precirculation embryonic tissues in vivo, and indicate that the only cells capable of adult long-term hematopoiesis are of intraembryonic origin.

Stress-induced ClpP serine protease of Listeria monocytogenes is essential for induction of listeriolysin O-dependent protective immunity.

The stress-induced protease ClpP is required for virulence of the facultative intracellular pathogen Listeria monocytogenes. We previously found that in the absence of ClpP, the virulence of this pathogen was strongly reduced, mainly due to the decreased production of functional listeriolysin O (LLO), a major immunodominant virulence factor promoting intracellular growth. In this work, a clpP deletion mutant of L. monocytogenes was used to study the generation of anti-Listeria protective immunity. We found that ClpP is required for the intracellular growth of L. monocytogenes in resident macrophages in vivo. Mice infected with doses as high as 10(6) clpP mutant bacteria were not protected against a lethal challenge of wild-type bacteria and did not develop any detectable LLO-specific cytolytic T cells or antibodies, suggesting that the amount of LLO produced in infected mice under these conditions was too low to induce a specific immune response. However, in contrast to the results obtained with a mutant with a disrupted hly gene, this lack of protection was overcome by inoculation of very high infecting doses of clpP mutant bacteria (5 x 10(8)), thus producing sufficient amounts of LLO to stimulate anti-Listeria immunity. The role of ClpP was confirmed by showing that anti-Listeria immunity was restored in mice infected with a clpP-complemented mutant. These results indicate that the stress-induced serine protease ClpP is a potential target for modulating the presentation of protective antigens such as LLO and thereby the immune response against L. monocytogenes.

Identification of committed NK cell progenitors in adult murine bone marrow.

Natural killer (NK) cells play important roles in innate immunity by lysing tumor and virally infected cells and by producing cytokines including interferon-gamma. While NK cell progenitors have been described in the fetal thymus, NK cell generation from hematopoietic stem cells (HSC) in the bone marrow (BM) occurs throughout life, and in athymic mice and humans. Interleukin (IL)-15 promotes NK development in vitro and is essential for the generation of normal numbers of NK cells in vivo. By characterizing BM cells expressing IL-15 receptor components, we found marked heterogeneity within the IL-2 receptor beta chain(+) (CD122(+)) subset, which included cells uniquely committed to the NK lineage. These CD122(+) NK cell precursors (NKP) are negative for markers used to identify mature NK cells, including NK1.1, DX5 and members of Ly-49 family, and fail to demonstrate natural cytotoxicity against susceptible target cells. In vitro culture of NKP generates mature lytic NK1.1(+) cells at high frequencies, while they do not give rise to T, B, myeloid or erythroid cells under appropriate conditions. NKP lack transcripts associated with early B and T cell differentiation (pTalpha, lambda5 and CD3epsilon), but express a group of genes (IL-15Ralpha, Id2, GATA-3 and Ets-1) and the 2B4 marker, which may define NK cell commitment. We propose that NKP represent the earliest adult BM precursor uniquely restricted to the NK cell lineage.

Functional dichotomy in natural killer cell signaling: Vav1-dependent and -independent mechanisms.

The product of the protooncogene Vav1 participates in multiple signaling pathways and is a critical regulator of antigen-receptor signaling in B and T lymphocytes, but its role during in vivo natural killer (NK) cell differentiation is not known. Here we have studied NK cell development in Vav1-/- mice and found that, in contrast to T and NK-T cells, the absolute numbers of phenotypically mature NK cells were not reduced. Vav1-/- mice produced normal amounts of interferon (IFN)-gamma in response to Listeria monocytogenes and controlled early infection but showed reduced tumor clearance in vivo. In vitro stimulation of surface receptors in Vav1-/- NK cells resulted in normal IFN-gamma production but reduced tumor cell lysis. Vav1 was found to control activation of extracellular signal-regulated kinases and exocytosis of cytotoxic granules. In contrast, conjugate formation appeared to be only mildly affected, and calcium mobilization was normal in Vav1-/- NK cells. These results highlight fundamental differences between proximal signaling events in T and NK cells and suggest a functional dichotomy for Vav1 in NK cells: a role in cytotoxicity but not for IFN-gamma production.

Natural killer cell-dependent apoptosis of peripheral murine hematopoietic progenitor cells in response to Fas cross-linking: involvement of tumor necrosis factor-alpha.

Recently, a marked extramedullary myelopoiesis in Fas/CD95- or FasL/CD95L-deficient mice has been reported. In the present in vitro study, the mechanisms underlying Fas-induced apoptosis of normal peripheral colony-forming unit-C (CFU-C) progenitors in the spleen were analyzed. Surprisingly, it was found that clonogenic progenitors were protected from gammaIFN plus Fas-induced programmed cell death when Lin(+) cells were removed from cultured splenocytes. The cells that rendered CFU-C sensitive to the activation of the Fas pathway did not belong to the T or the myelocytic-monocytic lineage but comprised a non-B-cell subset expressing the activation marker B220. Among CD19(-) B220(+) splenocytes, nearly half were natural killer (NK) 1.1(+) cells whose in vivo depletion or deficiency in RAG2-gamma(c)(-/-) mice abrogated the effect of Fas cross-linking. NK cells exerted their accessory function, at least in part, through tumor necrosis factor-alpha (TNF-alpha), which they readily produced during pretreatment with the anti-Fas/CD95 monoclonal antibody and IFN-gamma and whose addition could compensate for the loss of sensitivity. In conclusion, this study provides evidence that peripheral clonogenic progenitors are not directly responsive to Fas cross-linking, even in the presence of IFN-gamma, but require NK cells as a source of TNF-alpha to make them susceptible to this death pathway.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Differential requirement for the transcription factor PU.1 in the generation of natural killer cells versus B and T cells.

PU.1 is a member of the Ets family of transcription factors required for the development of various lymphoid and myeloid cell lineages, but its role in natural killer (NK) cell development is not known. The study shows that PU.1 is expressed in NK cells and that, on cell transfer into alymphoid Rag2/gammac(-/-) mice, hematopoietic progenitors of PU.1(-/-) fetal liver cells could generate functional NK cells but not B or T cells. Nevertheless, the numbers of bone marrow NK cell precursors and splenic mature NK cells were reduced compared to controls. Moreover, PU.1(-/-) NK cells displayed reduced expression of the receptors for stem cell factor and interleukin (IL)-7, suggesting a nonredundant role for PU.1 in regulating the expression of these cytokine receptor genes during NK cell development. PU.1(-/-) NK cells also showed defective expression of inhibitory and activating members of the Ly49 family and failed to proliferate in response to IL-2 and IL-12. Thus, despite the less stringent requirement for PU.1 in NK cell development compared to B and T cells, PU.1 regulates NK cell differentiation and homeostasis.

Inhibition of fibroblast growth factor/fibroblast growth factor receptor activity in glioma cells impedes tumor growth by both angiogenesis-dependent and -independent mechanisms.

We undertook a series of systematic studies to address the role of fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) activity in tumor growth and angiogenesis. We expressed dominant-negative FGFR2 (FGFR2-DN) or FGFR1 (FGFR1-DN) in glioma C6 cells by using constitutive or tetracycline-regulated expression systems. Anchorage-dependent or independent growth was inhibited in FGFR-DN-expressing cells. Tumor development after xenografting FGFR-DN-expressing cells in immunodeficient mice or after transplantation in rat brain was strongly inhibited. Quantification of microvessels demonstrated a significant decrease in vessel density in tumors derived from FGFR-DN-expressing cells. Furthermore, in a rabbit corneal assay, the angiogenic response after implantation of FGFR-DN-expressing cells was decreased. In tumors expressing FGFR-DN, vascular endothelial growth factor expression was strongly inhibited as compared with control tumor. These results indicate that inhibition of FGF activity may constitute a dominant therapeutic strategy in the treatment of FGF-producing cerebral malignancies and may disrupt both angiogenesis-dependent and -independent signals required for glioma growth and invasion.

Cytokines: IL-21 joins the gamma(c)-dependent network?

The discovery of the cytokine IL-21 adds another member to the ever-growing list of small secreted molecules that have potent effects on lymphoid cells. Initial characterization of the IL-21 receptor complex suggests that IL-21 may belong to the cytokine family whose receptors share the common gamma chain, gamma(c).