Fasting reveals largely intact systemic lipid mobilization mechanisms in respiratory chain complex III deficient mice

Mice homozygous for the human GRACILE syndrome mutation (Bcs1l^c.A232G) display decreased respiratory chain complex III activity, liver dysfunction, hypoglycemia, rapid loss of white adipose tissue and early death. To assess the underlying mechanism of the lipodystrophy in homozygous mice (Bcs1l^p.S78G), these and wild-type control mice were subjected to a short 4-hour fast. The homozygotes had low baseline blood glucose values, but a similar decrease in response to fasting as in wild-type mice, resulting in hypoglycemia in the majority. Despite the already depleted glycogen and increased triacylglycerol content in the mutant livers, the mice responded to fasting by further depletion and increase, respectively. Increased plasma free fatty acids (FAs) upon fasting suggested normal capacity for mobilization of lipids from white adipose tissue into circulation. Strikingly, however, serum glycerol concentration was not increased concomitantly with free FAs, suggesting its rapid uptake into the liver and utilization for fuel or gluconeogenesis in the mutants. The mutant hepatocyte mitochondria were capable of responding to fasting by appropriate morphological changes, as analyzed by electron microscopy, and by increasing respiration. Mutants showed increased hepatic gene expression of major metabolic controllers typically associated with fasting response (Ppargc1a, Fgf21, Cd36) already in the fed state, suggesting a chronic starvation-like metabolic condition. Despite this, the mutant mice responded largely normally to fasting by increasing hepatic respiration and switching to FA utilization, indicating that the mechanisms driving these adaptations are not compromised by the CIII dysfunction.Summary statementBcs1l mutant mice with severe CIII deficiency, energy deprivation and post-weaning lipolysis respond to fasting similarly to wild-type mice, suggesting largely normal systemic lipid mobilization and utilization mechanisms.     

Inactivation and Inducible Oncogenic Mutation of p53 in Gene Targeted Pigs

Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs) and live pigs carrying a latent TP53^R167H mutant allele, orthologous to oncogenic human mutant TP53^R175H and mouse Trp53^R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53^R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53^R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53^R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.     

Comparison of Effects of p53 Null and Gain-of-Function Mutations on Salivary Tumors in MMTV-Hras Transgenic Mice

p53 is an important tumor suppressor gene which is mutated in ~50% of all human cancers. Some of these mutants appear to have acquired novel functions beyond merely losing wild-type functions. To investigate these gain-of-function effects in vivo, we generated mice of three different genotypes: MMTV-Hras/p53^+/+, MMTV-Hras/p53^-/-, and MMTV-Hras/p53^R172H/R172H. Salivary tumors from these mice were characterized with regard to age of tumor onset, tumor growth rates, cell cycle distribution, apoptotic levels, tumor histopathology, as well as response to doxorubicin treatment. Microarray analysis was also performed to profile gene expression. The MMTV-Hras/p53^-/- and MMTV-Hras/p53^R172H/R172H mice displayed similar properties with regard to age of tumor onset, tumor growth rates, tumor histopathology, and response to doxorubicin, while both groups were clearly distinct from the MMTV-Hras/p53^+/+ mice by these measurements. In addition, the gene expression profiles of the MMTV-Hras/p53^-/- and MMTV-Hras/p53^R172H/R172H tumors were tightly clustered, and clearly distinct from the profiles of the MMTV-Hras/p53^+/+ tumors. Only a small group of genes showing differential expression between the MMTV-Hras/p53^-/- and MMTV-Hras/p53^R172H/R172H tumors, that did not appear to be regulated by wild-type p53, were identified. Taken together, these results indicate that in this MMTV-Hras-driven salivary tumor model, the major effect of the p53 R172H mutant is due to the loss of wild-type p53 function, with little or no gain-of-function effect on tumorigenesis, which may be explained by the tissue- and tumor type-specific properties of this gain-of-function mutant of p53.     

A novel nuclear interactor of ARF and MDM2 (NIAM) that maintains chromosomal stability

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.     

Loss of Function of the Mouse Sharpin Gene Results in Peyer’s Patch Regression

Peyer’s patches (PP) are an important component in the immune response against intestinal pathogens. Two independent, spontaneous mutations in the mouse Sharpin gene (Sharpin^cpdm and Sharpin^cpdm-Dem) result in the absence of PP and disrupted splenic white pulp in adult mice, although a full complement of lymph nodes is present. Here we report that rudimentary PP begin to develop in Sharpin^cpdm mice during embryogenesis, but lack the organizational patterns that are typical of this tissue. In the present study, small intestines examined at weekly intervals from birth to maturity showed spontaneous regression of PP in mutant mice with concurrent infiltration of granulocytes. At 5 to 6 weeks of age, only indistinct remnants of granulocytic accumulations remain. Transplantation of normal bone marrow into Sharpin^cpdm mice at 7 days of age did not prevent regression of PP in bone marrow chimeras examined at 7 to 8 weeks of age. These findings indicate that SHARPIN expression is required for the normal development and maintenance, but not initiation, of PP.     

Mutant p53R175H upregulates Twist1 expression and promotes epithelial–mesenchymal transition in immortalized prostate cells

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 ‘gain of function'' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53^R175H mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53^R175H, was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial–mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53^R175H mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53^R175H mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.     

The p53 tumour suppressor inhibits glucocorticoid-induced proliferation of erythroid progenitors

Hypoxia encountered at high altitude, blood loss and erythroleukemia instigate stress erythropoiesis, which involves glucocorticoid-induced proliferation of erythroid progenitors (ebls). The tumour suppressor p53 stimulates hematopoietic cell maturation and antagonizes glucocorticoid receptor (GR) activity in hypoxia, suggesting that it may inhibit stress erythropoiesis. We report that mouse fetal liver ebls that lack p53 proliferate better than wild-type cells in the presence of the GR agonist dexamethasone. An important mediator of GR-induced ebl self-renewal, the c-myb gene, is induced to higher levels in p53^–/– ebls by dexamethasone. The stress response to anemia is faster in the spleens of p53^–/– mice, as shown by the higher levels of colony forming units erythroids and the increase in the CD34/c-kit double positive population. Our results show that p53 antagonizes GR-mediated ebl expansion and demonstrate for the first time that p53–GR cross-talk is important in a physiological process in vivo: stress erythropoiesis.     

Visualizing Early Splenic Memory CD8+ T Cells Reactivation against Intracellular Bacteria in the Mouse

Memory CD8⁺ T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m). Memory CD8⁺ T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8⁺ T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs) fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m) as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8⁺ T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8⁺ T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8⁺ T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8⁺ T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8⁺ T produce inflammatory cytokines such as IFN-γ and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8⁺ T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8⁺ T cells provide a local response by secreting effector molecules around infected cells.     

Pten Null Prostate Epithelium Promotes Localized Myeloid-Derived Suppressor Cell Expansion and Immune Suppression during Tumor Initiation and Progression

Chronic inflammation is known to be associated with prostate cancer development, but how epithelium-associated cancer-initiating events cross talk to inflammatory cells during prostate cancer initiation and progression is largely unknown. Using the Pten null murine prostate cancer model, we show an expansion of Gr-1⁺ CD11b⁺ myeloid-derived suppressor cells (MDSCs) occurring intraprostatically immediately following epithelium-specific Pten deletion without expansion in hematopoietic tissues. This MDSC expansion is accompanied by sustained immune suppression. Prostatic Gr-1⁺ CD11b⁺ cells, but not those isolated from the spleen of the same tumor-bearing mice, suppress T cell proliferation and express high levels of Arginase 1 and iNOS. Mechanistically, the loss of PTEN in the epithelium leads to a significant upregulation of genes within the inflammatory response and cytokine-cytokine receptor interaction pathways, including Csf1 and Il1b, two genes known to induce MDSC expansion and immunosuppressive activities. Treatment of Pten null mice with the selective CSF-1 receptor inhibitor GW2580 decreases MDSC infiltration and relieves the associated immunosuppressive phenotype. Our study indicates that epithelium-associated tumor-initiating events trigger the secretion of inflammatory cytokines and promote localized MDSC expansion and immune suppression, thereby promoting tumor progression.     

Loss of PML cooperates with mutant p53 to drive more aggressive cancers in a gender-dependent manner

p53 mutations and downregulation of promyelocytic leukemia (PML) are common genetic alterations in human cancers. In healthy cells these two key tumor suppressors exist in a positive regulatory loop, promoting cell death and cellular senescence. However, the influence of their interplay on tumorigenesis has not been explored directly in vivo. The contribution of PML to mutant p53 driven cancer was evaluated in a mouse model harboring a p53 mutation (p53^{wild-type/R172H}) that recapitulates a frequent p53 mutation (p53^R175H) in human sporadic and Li-Fraumeni cancers. These mice with PML displayed perturbation of the hematopoietic compartment, manifested either as lymphoma or extramedullary hematopoiesis (EMH). EMH was associated with peripheral blood leucocytosis and macrocytic anemia, suggestive of myeloproliferative- myelodysplastic overlap. In contrast, a complete loss of PML from these mice resulted in a marked alteration in tumor profile. While the incidence of lymphomas was unaltered, EMH was not detected and the majority of mice succumbed to sarcomas. Further, males lacking PML exhibited a high incidence of soft tissue sarcomas and reduced survival, while females largely developed osteosarcomas, without impact on survival. Together, these findings demonstrate that PML is an important tumor suppressor dictating disease development in a pertinent mouse model of human cancer.

Key Points: (1) A mutant p53 allele disrupts hematopoiesis in mice, by promoting lymphomas and myeloproliferative / myelodysplastic overlap. (2) Coincidental p53 allele mutation and PML loss shifts the tumor profile toward sarcoma formation, which is paralleled in human leiomyosarcomas (indicated by immunohistochemistry; IHC).     

Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity,enhanced immunosuppression and cellular senescence

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53^R172P embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV-hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53^R172P MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.Key words: UVB irradiation, p53, DNA damage, DNA damage responses, apoptosis, senescence     

The Neurotrophic Receptor Ntrk2 Directs Lymphoid Tissue Neovascularization during Leishmania donovani Infection

The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80^hiCD11b^loCD11c⁺ macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis.     

Infection with Salmonella enterica Serovar Typhimurium Leads to Increased Proportions of F4/80+ Red Pulp Macrophages and Decreased Proportions of B and T Lymphocytes in the Spleen

Infection of mice with Salmonella enterica serovar Typhimurium (Salmonella) causes systemic inflammatory disease and enlargement of the spleen (splenomegaly). Splenomegaly has been attributed to a general increase in the numbers of phagocytes, lymphocytes, as well as to the expansion of immature CD71⁺Ter119⁺ reticulocytes. The spleen is important for recycling senescent red blood cells (RBCs) and for the capture and eradication of blood-borne pathogens. Conservation of splenic tissue architecture, comprised of the white pulp (WP), marginal zone (MZ), and red pulp (RP) is essential for initiation of adaptive immune responses to captured pathogens. Using flow cytometry and four color immunofluorescence microscopy (IFM), we show that Salmonella-induced splenomegaly is characterized by drastic alterations of the splenic tissue architecture and cell population proportions, as well as in situ cell distributions. A major cause of splenomegaly appears to be the significant increase in immature RBC precursors and F4/80⁺ macrophages that are important for recycling of heme-associated iron. In contrast, the proportions of B220⁺, CD4⁺ and CD8⁺ lymphocytes, as well as MZ MOMA⁺ macrophages decrease significantly as infection progresses. Spleen tissue sections show visible tears and significantly altered tissue architecture with F4/80⁺ macrophages and RBCs expanding beyond the RP and taking over most of the spleen tissue. Additionally, F4/80⁺ macrophages actively phagocytose not only RBCs, but also lymphocytes, indicating that they may contribute to declining lymphocyte proportions during Salmonella infection. Understanding how these alterations of spleen microarchitecture impact the generation of adaptive immune responses to Salmonella has implications for understanding Salmonella pathogenesis and for the design of more effective Salmonella-based vaccines.     

Effect of Bcl11b genotypes and gamma-radiation on the development of mouse thymic lymphomas

Bcl11b is a haploinsufficient tumor suppressor gene and expressed in many tissues such as thymus, brain and skin. Irradiated Bcl11b^+/− heterozygous mice mostly develop thymic lymphomas, but the preference of Bcl11b inactivation for thymic lymphomas remains to be addressed. We produced Bcl11b^+/− heterozygous and Bcl11b wild-type mice of p53^+/− background and compared their incidence of γ-ray induced thymic lymphomas. Majority of the tumors in p53^+/− mice were skin tumors, and only 5 (36%) of the 14 tumors were thymic lymphomas. In contrast, Bcl11b^+/−p53^+/− doubly heterozygous mice developed thymic lymphomas at the frequency of 27 (79%) of the 34 tumors developed (P = 0.008). This indicates the preference of Bcl11b impairment for thymic lymphoma development. We also analyzed loss of the wild-type alleles in the 27 lymphomas, a predicted consequence given by γ-irradiation. However, the loss frequency was low, only six (22%) for Bcl11b and five (19%) for p53. The frequencies did not differ from those of spontaneously developed thymic lymphomas in the doubly heterozygous mice, though the latency of lymphoma development markedly differed between them. This suggests that the main contribution of irradiation at least in those mice is not for the tumor initiation by inducing allelic losses but probably for the promotion of thymic lymphoma development.     

CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection

During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8⁺ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8⁺ T cells recognize the epitope YopE_69-77. The features of the interaction between pathogen and host that result in this large CD8⁺ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE_69-77-specific CD8⁺ T cells generated during the large response are polyclonal and are produced by a “translocation-dependent” pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE_69-77-specific CD8⁺ T cell response (~10% of the large expansion) can be generated in a “translocation-independent” pathway in which CD8α⁺ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE_69-77-specific CD8⁺ T cell expansion because this response was significantly reduced in Ccr2^-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE_69-77-specific CD8⁺ T cells ex vivo and promoted the expansion of YopE_69-77-specific CD8⁺ T cells in infected Ccr2^-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8⁺ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE_69-77-specific CD8⁺ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.