DIDO is necessary for the adipogenesis that promotes diet-induced obesity.

The prevalence of overweight and obesity continues to rise in the population worldwide. Because it is an important predisposing factor for cancer, cardiovascular diseases, diabetes mellitus, and COVID-19, obesity reduces life expectancy. Adipose tissue (AT), the main fat storage organ with endocrine capacity, plays fundamental roles in systemic metabolism and obesity-related diseases. Dysfunctional AT can induce excess or reduced body fat (lipodystrophy). Dido1 is a marker gene for stemness; gene-targeting experiments compromised several functions ranging from cell division to embryonic stem cell differentiation, both in vivo and in vitro. We report that mutant mice lacking the DIDO N terminus show a lean phenotype. This consists of reduced AT and hypolipidemia, even when mice are fed a high-nutrient diet. DIDO mutation caused hypothermia due to lipoatrophy of white adipose tissue (WAT) and dermal fat thinning. Deep sequencing of the epididymal white fat (Epi WAT) transcriptome supported Dido1 control of the cellular lipid metabolic process. We found that, by controlling the expression of transcription factors such as C/EBPα or PPARγ, Dido1 is necessary for adipocyte differentiation, and that restoring their expression reestablished adipogenesis capacity in Dido1 mutants. Our model differs from other lipodystrophic mice and could constitute a new system for the development of therapeutic intervention in obesity.

Growth Hormone Reprograms Macrophages toward an Anti-Inflammatory and Reparative Profile in an MAFB-Dependent Manner.

Growth hormone (GH), a pleiotropic hormone secreted by the pituitary gland, regulates immune and inflammatory responses. In this study, we show that GH regulates the phenotypic and functional plasticity of macrophages both in vitro and in vivo. Specifically, GH treatment of GM-CSF-primed monocyte-derived macrophages promotes a significant enrichment of anti-inflammatory genes and dampens the proinflammatory cytokine profile through PI3K-mediated downregulation of activin A and upregulation of MAFB, a critical transcription factor for anti-inflammatory polarization of human macrophages. These in vitro data correlate with improved remission of inflammation and mucosal repair during recovery in the acute dextran sodium sulfate-induced colitis model in GH-overexpressing mice. In this model, in addition to the GH-mediated effects on other immune cells, we observed that macrophages from inflamed gut acquire an anti-inflammatory/reparative profile. Overall, these data indicate that GH reprograms inflammatory macrophages to an anti-inflammatory phenotype and improves resolution during pathologic inflammatory responses.

Role of Innate and Adaptive Cytokines in the Survival of COVID-19 Patients.

SARS-CoV-2 is a new coronavirus characterized by a high infection and transmission capacity. A significant number of patients develop inadequate immune responses that produce massive releases of cytokines that compromise their survival. Soluble factors are clinically and pathologically relevant in COVID-19 survival but remain only partially characterized. The objective of this work was to simultaneously study 62 circulating soluble factors, including innate and adaptive cytokines and their soluble receptors, chemokines and growth and wound-healing/repair factors, in severe COVID-19 patients who survived compared to those with fatal outcomes. Serum samples were obtained from 286 COVID-19 patients and 40 healthy controls. The 62 circulating soluble factors were quantified using a Luminex Milliplex assay. Results. The patients who survived had decreased levels of the following 30 soluble factors of the 62 studied compared to those with fatal outcomes, therefore, these decreases were observed for cytokines and receptors predominantly produced by the innate immune system-IL-1α, IL-1α, IL-18, IL-15, IL-12p40, IL-6, IL-27, IL-1Ra, IL-1RI, IL-1RII, TNFα, TGFα, IL-10, sRAGE, sTNF-RI and sTNF-RII-for the chemokines IL-8, IP-10, MCP-1, MCP-3, MIG and fractalkine; for the growth factors M-CSF and the soluble receptor sIL2Ra; for the cytokines involved in the adaptive immune system IFNγ, IL-17 and sIL-4R; and for the wound-repair factor FGF2. On the other hand, the patients who survived had elevated levels of the soluble factors TNFß, sCD40L, MDC, RANTES, G-CSF, GM-CSF, EGF, PDGFAA and PDGFABBB compared to those who died. Conclusions. Increases in the circulating levels of the sCD40L cytokine; MDC and RANTES chemokines; the G-CSF and GM-CSF growth factors, EGF, PDGFAA and PDGFABBB; and tissue-repair factors are strongly associated with survival. By contrast, large increases in IL-15, IL-6, IL-18, IL-27 and IL-10; the sIL-1RI, sIL1RII and sTNF-RII receptors; the MCP3, IL-8, MIG and IP-10 chemokines; the M-CSF and sIL-2Ra growth factors; and the wound-healing factor FGF2 favor fatal outcomes of the disease.

Dido3-dependent SFPQ recruitment maintains efficiency in mammalian alternative splicing.

Alternative splicing is facilitated by accessory proteins that guide spliceosome subunits to the primary transcript. Many of these splicing factors recognize the RNA polymerase II tail, but SFPQ is a notable exception even though essential for mammalian RNA processing. This study reveals a novel role for Dido3, one of three Dido gene products, in alternative splicing. Binding of the Dido3 amino terminus to histones and to the polymerase jaw domain was previously reported, and here we show interaction between its carboxy terminus and SFPQ. We generated a mutant that eliminates Dido3 but preserves other Dido gene products, mimicking reduced Dido3 levels in myeloid neoplasms. Dido mutation suppressed SFPQ binding to RNA and increased skipping for a large group of exons. Exons bearing recognition sequences for alternative splicing factors were nonetheless included more efficiently. Reduced SFPQ recruitment may thus account for increased skipping of SFPQ-dependent exons, but could also generate a splicing factor surplus that becomes available to competing splice sites. Taken together, our data indicate that Dido3 is an adaptor that controls SFPQ utilization in RNA splicing. Distributing splicing factor recruitment over parallel pathways provides mammals with a simple mechanism to regulate exon usage while maintaining RNA splicing efficiency.

Dido mutations trigger perinatal death and generate brain abnormalities and behavioral alterations in surviving adult mice.

Nearly all vertebrate cells have a single cilium protruding from their surface. This threadlike organelle, once considered vestigial, is now seen as a pivotal element for detection of extracellular signals that trigger crucial morphogenetic pathways. We recently proposed a role for Dido3, the main product of the death inducer-obliterator (dido) gene, in histone deacetylase 6 delivery to the primary cilium [Sánchez de Diego A, et al. (2014) Nat Commun 5:3500]. Here we used mice that express truncated forms of Dido proteins to determine the link with cilium-associated disorders. We describe dido mutant mice with high incidence of perinatal lethality and distinct neurodevelopmental, morphogenetic, and metabolic alterations. The anatomical abnormalities were related to brain and orofacial development, consistent with the known roles of primary cilia in brain patterning, hydrocephalus incidence, and cleft palate. Mutant mice that reached adulthood showed reduced life expectancy, brain malformations including hippocampus hypoplasia and agenesis of corpus callosum, as well as neuromuscular and behavioral alterations. These mice can be considered a model for the study of ciliopathies and provide information for assessing diagnosis and therapy of genetic disorders linked to the deregulation of primary cilia.

p38α regulates cytokine-induced IFNγ secretion via the Mnk1/eIF4E pathway in Th1 cells.

The p38 mitogen-activated protein kinase (MAPK) pathway is involved in the regulation of immune and inflammatory processes. We used p38α-conditional, p38ß-deficient and p38α/ß double-null mouse models to address the role of these two p38 MAPK in CD4+ T cells, and found that p38α deficiency causes these cells to hyperproliferate. Our studies indicate that both p38α and p38ß are dispensable for T helper cell type 1 (Th1) differentiation but, by controlling interferon (IFN)γ and tumor necrosis factor (TNF)α production, are critical for normal Th1 effector function. We found that both p38α and p38ß modulate T-cell receptor-induced IFNγ and TNFα production, whereas only p38α regulates cytokine-induced IFNγ production. The lack of p38α and p38ß did not affect transcription and mRNA stability of Ifng. However, the absence of p38α in Th1 cells resulted in a decreased MNK1 phosphorylation after cytokine activation, and MNK1 inhibition blocked IFNγ production. Our results indicate that p38α regulates IFNγ secretion through the activation of the MNK1/eIF4E pathway of translation initiation and identify specific functions for p38α and p38ß in T-cell proliferation.

The expression of Death Inducer-Obliterator (DIDO) variants in Myeloproliferative Neoplasms.

Chronic Myeloid Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation without cell maturation impairment. CML pathogenesis is associated with the Ph chromosome leading to BCR-ABL tyrosine-kinase constitutive expression. The Ph negative MPN (PV, ET and PMF) are characterized by the mutation JAK2(V617F) of the JAK2 protein in the auto-inhibitory JH2 domain, which is found in most PV patients and in approximately half of ET and PMF patients. Considerable effort is being made to understand the role of JAK2(V617F) at the MPN initiation and to clarify the pathogenesis and apoptosis resistance in CML, PV, ET and PMF patients. In the present investigation, we evaluated the Death Inducer-Obliterator (DIDO) (variants DIDO 1, 2 and 3) levels in CML, PV, ET and PMF patients. Our data reported the DIDO 1, 2 and 3 differential expressions in Myeloproliferative Neoplasms.

Growth hormone prevents the development of autoimmune diabetes.

Evidence supports a relationship between the neuroendocrine and the immune systems. Data from mice that overexpress or are deficient in growth hormone (GH) indicate that GH stimulates T and B-cell proliferation and Ig synthesis, and enhances maturation of myeloid progenitor cells. The effect of GH on autoimmune pathologies has nonetheless been little studied. Using a murine model of type 1 diabetes, a T-cell-mediated autoimmune disease characterized by immune cell infiltration of pancreatic islets and destruction of insulin-producing ß-cells, we observed that sustained GH expression reduced prodromal disease symptoms and eliminated progression to overt diabetes. The effect involves several GH-mediated mechanisms; GH altered the cytokine environment, triggered anti-inflammatory macrophage (M2) polarization, maintained activity of the suppressor T-cell population, and limited Th17 cell plasticity. In addition, GH reduced apoptosis and/or increased the proliferative rate of ß-cells. These results support a role for GH in immune response regulation and identify a unique target for therapeutic intervention in type 1 diabetes.

Pathogens: raft hijackers.

Throughout evolution, organisms have developed immune-surveillance networks to protect themselves from potential pathogens. At the cellular level, the signalling events that regulate these defensive responses take place in membrane rafts--dynamic microdomains that are enriched in cholesterol and glycosphingolipids--that facilitate many protein-protein and lipid-protein interactions at the cell surface. Pathogens have evolved many strategies to ensure their own survival and to evade the host immune system, in some cases by hijacking rafts. However, understanding the means by which pathogens exploit rafts might lead to new therapeutic strategies to prevent or alleviate certain infectious diseases, such as those caused by HIV-1 or Ebola virus.

Abnormal chemokine receptor profile on circulating T lymphocytes from nonallergic asthma patients.

BACKGROUND: T lymphocytes are involved in the pathogenesis of nonallergic asthma. The objective of this study was to characterize the subset distribution and pattern of chemokine receptor expression in circulating T lymphocyte subsets from nonallergic asthma patients. METHODS: Forty stable nonallergic asthma patients and 16 sex- and age-matched healthy donors were studied. Twelve patients did not receive inhaled steroids (untreated patients), 16 received 50-500 µg b.i.d. of inhaled fluticasone propionate (FP) (standard-dose patients), and 12 received over 500 µg b.i.d. of inhaled FP (high-dose patients) for at least 12 months prior to the beginning of this study and were clinically well controlled. Flow cytometry was performed using a panel of monoclonal antibodies (4 colors). RESULTS: Nonallergic asthma patients treated with high doses of inhaled FP showed a significant reduction in the percentages of CD3+ T lymphocytes compared to healthy controls. Untreated patients showed a significant increase in CCR6 expression in CD8+CD25+ and CD8+CD25+bright T cells compared to healthy controls. The results were similar for CXCR3 and CCR5 expression. In patients treated with standard doses of FP, CCR5 expression was significantly increased in CD3+ T lymphocytes relative to healthy controls. CONCLUSIONS: The different groups of clinically stable nonallergic asthmatic patients showed distinct patterns of alterations in subset distribution as well as CCR6, CXCR3, and CCR5 expression on circulating T lymphocytes. .

Filamin-A regulates actin-dependent clustering of HIV receptors.

Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA-ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infection.

PARP1, DIDO3, and DHX9 Proteins Mutually Interact in Mouse Fibroblasts, with Effects on DNA Replication Dynamics, Senescence, and Oncogenic Transformation.

The regulated formation and resolution of R-loops is a natural process in physiological gene expression. Defects in R-loop metabolism can lead to DNA replication stress, which is associated with a variety of diseases and, ultimately, with cancer. The proteins PARP1, DIDO3, and DHX9 are important players in R-loop regulation. We previously described the interaction between DIDO3 and DHX9. Here, we show that, in mouse embryonic fibroblasts, the three proteins are physically linked and dependent on PARP1 activity. The C-terminal truncation of DIDO3 leads to the impairment of this interaction; concomitantly, the cells show increased replication stress and senescence. DIDO3 truncation also renders the cells partially resistant to in vitro oncogenic transformation, an effect that can be reversed by immortalization. We propose that PARP1, DIDO3, and DHX9 proteins form a ternary complex that regulates R-loop metabolism, preventing DNA replication stress and subsequent senescence.

Notch activation stimulates migration of breast cancer cells and promotes tumor growth.

INTRODUCTION: Dysregulated NOTCH receptor activity has been implicated in breast cancer but the mechanisms by which NOTCH contributes to transformation are not yet clear, as it has context-dependent effects on the properties of transformed cells. METHODS: We have used various in vitro and in vivo carcinogenic models to analyze the impact of Notch signaling in the onset and progression of breast tumors. RESULTS: We found that ectopic expression of the Notch1 intracellular domain (N1ICD) in MCF-7 breast adenocarcinoma cell line caused reduction and delocalization of E-CADHERIN levels and increased migratory and invasive abilities. Notch inhibition in the invasive breast cancer cell line MDA-MB-231 resulted in increased E-CADHERIN expression and a parallel reduction in their invasive capacity. The growth of subcutaneous xenografts produced with MCF-7 cells was boosted after N1ICD induction, in a cell autonomous manner. In vivo Notch1 activation in the mammary gland using the MMTV-Cre driver caused the formation of papillary tumors that showed increased Hes1 and Hey1 expression and delocalized E-cadherin staining. CONCLUSIONS: These results confirm NOTCH1 as a signal triggering epithelial-mesenchymal transition in epithelial cancer cells, which may have implications in tumor dissemination, metastasis and proliferation in vivo. The identification of specific factors interacting with NOTCH signaling could thus be relevant to fully understanding the role of NOTCH in breast neoplasia.

A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress.

Recent observations using multiphoton intravital microscopy (MP-IVM) have uncovered an unexpectedly high lymphocyte motility within peripheral lymph nodes (PLNs). Lymphocyte-expressed intracellular signaling molecules governing interstitial movement remain largely unknown. Here, we used MP-IVM of murine PLNs to examine interstitial motility of lymphocytes lacking the Rac guanine exchange factor DOCK2 and phosphoinositide-3-kinase (PI3K)gamma, signaling molecules that act downstream of G protein-coupled receptors, including chemokine receptors (CKRs). T and B cells lacking DOCK2 alone or DOCK2 and PI3Kgamma displayed markedly reduced motility inside T cell area and B cell follicle, respectively. Lack of PI3Kgamma alone had no effect on migration velocity but resulted in increased turning angles of T cells. As lymphocyte egress from PLNs requires the sphingosine-1-phosphate (S1P) receptor 1, a G(alphai) protein-coupled receptor similar to CKR, we further analyzed whether DOCK2 and PI3Kgamma contributed to S1P-triggered signaling events. S1P-induced cell migration was significantly reduced in T and B cells lacking DOCK2, whereas T cell-expressed PI3Kgamma contributed to F-actin polymerization and protein kinase B phosphorylation but not migration. These findings correlated with delayed lymphocyte egress from PLNs in the absence of DOCK2 but not PI3Kgamma, and a markedly reduced cell motility of DOCK2-deficient T cells in close proximity to efferent lymphatic vessels. In summary, our data support a central role for DOCK2, and to a lesser extent T cell-expressed PI3Kgamma, for signal transduction during interstitial lymphocyte migration and S1P-mediated egress.

Centromere-localized breaks indicate the generation of DNA damage by the mitotic spindle.

Most carcinomas present some form of chromosome instability in combination with spindle defects. Numerical instability is likely caused by spindle aberrations, but the origin of breaks and translocations remains elusive. To determine whether one mechanism can bring about both types of instability, we studied the relationship between DNA damage and spindle defects. Although lacking apparent repair defects, primary Dido mutant cells formed micronuclei containing damaged DNA. The presence of centromeres showed that micronuclei were caused by spindle defects, and cell cycle markers showed that DNA damage was generated during mitosis. Although the micronuclei themselves persisted, the DNA damage within was repaired during S and G2 phases. DNA breaks in Dido mutant cells regularly colocalized with centromeres, which were occasionally distorted. Comparable defects were found in APC mutant cell lines, an independent system for spindle defects. On the basis of these results, we propose a model for break formation in which spindle defects lead to centromere shearing.

Tyr³²³-dependent p38 activation is associated with rheumatoid arthritis and correlates with disease activity.

OBJECTIVE: The p38 MAPK is important in the pathogenic immune response in rheumatoid arthritis (RA). The p38 molecule can be activated through phosphorylation on Thr¹8°-Tyr¹8² by upstream MAPK kinases and via an alternative pathway through phosphorylation on Tyr³²³. We undertook this study to quantify the phosphorylation of Tyr³²³ p38 and of Thr¹8°-Tyr¹8² p38 on T cells from healthy controls and patients with RA or ankylosing spondylitis (AS) to identify variables associated with p38 phosphorylation and disease activity. METHODS: We measured p38 phosphorylation on Tyr³²³ and Thr¹8°-Tyr¹8² by flow cytometry and Western blotting on T cells from 30 control subjects, 33 AS patients, 30 patients with RA in remission, and 79 patients with active RA. We collected the clinical characteristics and analyzed correlations between clinical variables, the Disease Activity Score in 28 joints (DAS28), and p38 phosphorylation levels. Multivariate regression analysis was performed to identify variables associated with p38 phosphorylation on Tyr³²³ and Thr¹8°-Tyr¹8². RESULTS: Phosphorylation of p38 on Tyr³²³ was higher in T cells from patients with active RA (P = 0.008 versus healthy controls) than in patients with RA in remission or in patients with AS. Tyr³²³ p38 phosphorylation was associated with disease activity determined by the DAS28 (P = 0.017). Enhanced p38 phosphorylation was linked to Lck-mediated activation of the Tyr³²³-dependent pathway in the absence of upstream MAPKK activation. CONCLUSION: Our results indicate that phosphorylation status on Tyr³²³ p38 correlates with RA disease activity and suggest that the Tyr³²³-dependent pathway is an attractive target for down-regulation of p38 activity in RA patients.

PI3Kgamma inhibition blocks glomerulonephritis and extends lifespan in a mouse model of systemic lupus.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease generated by deregulation of T cell-mediated B-cell activation, which results in glomerulonephritis and renal failure. Disease is treated with immunosuppressants and cytostatic agents that have numerous side effects. Here we examine the use of inhibitors of phosphoinositide 3-kinase (PI3K) gamma, a lipid kinase that regulates inflammation, in the MRL-lpr mouse model of SLE. Treatment reduced glomerulonephritis and prolonged lifespan, suggesting that P13Kgamma may be a useful target in the treatment of chronic inflammation.

APRIL promotes B-1 cell-associated neoplasm.

A tumor-supporting role for the TNF-like ligand APRIL has been suggested. Here we describe that 9- to 12-month-old APRIL transgenic mice develop lymphoid tumors that originate from expansion of the peritoneal B-1 B cell population. Aging APRIL transgenic mice develop progressive hyperplasia in mesenteric lymph nodes and Peyer's patches, disorganization of affected lymphoid tissues, mucosal and capsular infiltration, and eventual tumor cell infiltration into nonlymphoid tissues such as kidney and liver. We detected significantly increased APRIL levels in sera of B cell chronic lymphoid leukemia (B-CLL) patients, indicating that APRIL promotes onset of B-1-associated neoplasms and that APRIL antagonism may provide a therapeutic strategy to treat B-CLL patients.

Hepatitis, testicular degeneration, and ataxia in DIDO3-deficient mice with altered mRNA processing.

BACKGROUND: mRNA processing is an essential step of gene expression; its malfunction can lead to different degrees of physiological disorder from subclinical disease to death. We previously identified Dido1 as a stemness marker and a gene involved in embryonic stem cell differentiation. DIDO3, the largest protein encoded by the Dido1 gene, is necessary for accurate mRNA splicing and correct transcription termination. The deletion of Dido1 exon16, which encodes the carboxy-terminal half of DIDO3, results in early embryonic lethality in mouse. RESULTS: We obtained mice bearing a Cre-LoxP conditional version of that deletion and studied the effects of inducing it ubiquitously in adult stages. DIDO3-deficient mice survive the deletion but suffer mild hepatitis, testicular degeneration, and progressive ataxia, in association with systemic alterations in mRNA splicing and transcriptional readthrough. CONCLUSIONS: These results offer insight into the distinct vulnerabilities in mouse organs following impairment of the mRNA processing machinery, and could aid understanding of human health dependence on accurate mRNA metabolism.