Inducible and reversible inhibition of mirna-mediated gene repression in vivo

Although virtually all gene networks are predicted to be controlled by miRNAs, the contribution of this important layer of gene regulation to tissue homeostasis in adult animals remains unclear. Gain and loss of function experiments have provided key insights into the specific function of individual miRNAs, but effective genetic tools to study the functional consequences of global inhibition of miRNA activity in vivo are lacking. Here we report the generation and characterization of a genetically engineered mouse strain in which miRNA-mediated gene repression can be reversibly inhibited without affecting miRNA biogenesis or abundance. We demonstrate the usefulness of this strategy by investigating the consequences of acute inhibition of miRNA function in adult animals. We find that different tissues and organs respond differently to global loss of miRNA function. While miRNA-mediated gene repression is essential for the homeostasis of the heart and the skeletal muscle, it is largely dispensable in the majority of other organs. Even in tissues where it is not required for homeostasis, such as the intestine and hematopoietic system, miRNA activity can become essential during regeneration following acute injury. These data support a model where many metazoan tissues primarily rely on miRNA function to respond to potentially pathogenic events

Conformational changes and protein stability of the pro-apoptotic protein Bax

Pro-apoptotic Bax is a soluble and monomeric protein under normal physiological conditions. Upon its activation substantial structural rearrangements occur: The protein inserts into the mitochondrial outer membrane and forms higher molecular weight oligomers. Subsequently, the cells can undergo apoptosis. In our studies, we focused on the structural rearrangements of Bax during oligomerization and on the protein stability. Both protein conformations exhibit high stability against thermal denaturation, chemically induced unfolding and proteolytic processing. The oligomeric protein is stable up to 90 °C as well as in solutions of 8 M urea or 6 M guanidinium hydrochloride. Helix 9 appears accessible in the monomer but hidden in the oligomer assessed by proteolysis. Tryptophan fluorescence indicates that the environment of the C-terminal protein half becomes more apolar upon oligomerization, whereas the loop region between helices 1 and 2 gets solvent exposed

Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis

The process of apoptosis in immune cells like mast cells is essential to regain homeostasis after an inflammatory response. The intrinsic pathway of apoptosis is ultimately controlled by the pro-apoptotic Bcl-2 family members Bax and Bak, which upon activation oligomerize to cause increased permeabilization of the mitochondria outer membrane leading to cell death. We examined the role of Bax and Bak in cytokine deprivation-induced apoptosis in mast cells using connective tissue-like mast cells and mucosal-like mast cells derived from bax−/−, bak−/− and bax−/−bak−/− mice. Although both Bax and Bak were expressed at readily detectable protein levels, we found a major role for Bax in mediating mast cell apoptosis induced by cytokine deprivation. We analyzed cell viability by propidium iodide exclusion and flow cytometry after deprivation of vital cytokines for each mast cell population. Upon cytokine withdrawal, bak−/− mast cells died at a similar rate as wild type, whereas bax−/− and bax−/−bak−/− mast cells were partially or completely resistant to apoptosis, respectively. The total resistance seen in bax−/−bak−/− mast cells is comparable with mast cells deficient of both pro-apoptotic Bim and Puma or mast cells overexpressing anti-apoptotic Bcl-2. These results show that Bax has a predominant and Bak a minor role in cytokine deprivation-induced apoptosis in both connective tissue-like and mucosal-like mast cells

Mycobacterium tuberculosis Induces an Atypical Cell Death Mode to Escape from Infected Macrophages

BACKGROUND: Macrophage cell death following infection with Mycobacterium tuberculosis plays a central role in tuberculosis disease pathogenesis. Certain attenuated strains induce extrinsic apoptosis of infected macrophages but virulent strains of M. tuberculosis suppress this host response. We previously reported that virulent M. tuberculosis induces cell death when bacillary load exceeds approximately 20 per macrophage but the precise nature of this demise has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the characteristics of cell death in primary murine macrophages challenged with virulent or attenuated M. tuberculosis complex strains. We report that high intracellular bacillary burden causes rapid and primarily necrotic death via lysosomal permeabilization, releasing hydrolases that promote Bax/Bak-independent mitochondrial damage and necrosis. Cell death was independent of cathepsins B or L and notable for ultrastructural evidence of damage to lipid bilayers throughout host cells with depletion of several host phospholipid species. These events require viable bacteria that can respond to intracellular cues via the PhoPR sensor kinase system but are independent of the ESX1 system. CONCLUSIONS/SIGNIFICANCE: Cell death caused by virulent M. tuberculosis is distinct from classical apoptosis, pyroptosis or pyronecrosis. Mycobacterial genes essential for cytotoxicity are regulated by the PhoPR two-component system. This atypical death mode provides a mechanism for viable bacilli to exit host macrophages for spreading infection and the eventual transition to extracellular persistence that characterizes advanced pulmonary tuberculosis

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

Glucose Induces Pancreatic Islet Cell Apoptosis That Requires the BH3-Only Proteins Bim and Puma and Multi-BH Domain Protein Bax

OBJECTIVE: High concentrations of circulating glucose are believed to contribute to defective insulin secretion and beta-cell function in diabetes and at least some of this effect appears to be caused by glucose-induced beta-cell apoptosis. In mammalian cells, apoptotic cell death is controlled by the interplay of proapoptotic and antiapoptotic members of the Bcl-2 family. We investigated the apoptotic pathway induced in mouse pancreatic islet cells after exposure to high concentrations of the reducing sugars ribose and glucose as a model of beta-cell death due to long-term metabolic stress. RESEARCH DESIGN AND METHODS: Islets isolated from mice lacking molecules implicated in cell death pathways were exposed to high concentrations of glucose or ribose. Apoptosis was measured by analysis of DNA fragmentation and release of mitochondrial cytochrome c. RESULTS: Deficiency of interleukin-1 receptors or Fas did not diminish apoptosis, making involvement of inflammatory cytokine receptor or death receptor signaling in glucose-induced apoptosis unlikely. In contrast, overexpression of the prosurvival protein Bcl-2 or deficiency of the apoptosis initiating BH3-only proteins Bim or Puma, or the downstream apoptosis effector Bax, markedly reduced glucose- or ribose-induced killing of islets. Loss of other BH3-only proteins Bid or Noxa, or the Bax-related effector Bak, had no impact on glucose-induced apoptosis. CONCLUSIONS: These results implicate the Bcl-2 regulated apoptotic pathway in glucose-induced islet cell killing and indicate points in the pathway at which interventional strategies can be designed

Cytokine-dependent and cytokine-independent roles for Mcl-1: genetic evidence for multiple mechanisms by which Mcl-1 promotes survival in primary T lymphocytes

Myeloid cell leukemia sequence-1 (Mcl-1) is a critical anti-apoptotic factor in T lymphocytes. However, in spite of the many pro-apoptotic proteins with proposed binding to Mcl-1, the specific interactions by which Mcl-1 regulates primary T-cell survival under different conditions have not been fully explored. Further, how different trophic cytokines modulate the specific role(s) of Mcl-1 is unknown. Here, we use genetic mouse models to dissect the roles of Mcl-1 in primary T lymphocytes. Using the inducible Mcl-1-floxed estrogen receptor-Cre fusion protein (Mcl-1f/fERCre) deletion system in combination with genetic modification of other B-cell lymphoma 2 (Bcl-2) family members, we show that loss of pro-apoptotic Bcl-2 homologous antagonist/killer (Bak) rescues the survival of Mcl-1-deficient T cells in the presence of IL-7. Without IL-7, the survival of Mcl-1-deficient cells cannot be rescued by loss of Bak, but is partially rescued by overexpression of Bcl-2 or loss of Bcl-2-interacting mediator of cell death (Bim). Thus, Mcl-1 and Bcl-2 have a shared role, the inhibition of Bim, in promoting T-cell survival during cytokine withdrawal. Finally, we show that other common gamma-chain (γc) cytokines differentially modulate the roles of Mcl-1. IL-15 has effects similar to those of IL-7 in memory T cells and naïve CD8+ cells, but not naïve CD4+ cells. However, IL-4 maintains Mcl-1 and Bcl-2 but also upregulates Bim and Bcl-2-associated X protein (Bax), thus altering the cell's dependence on Mcl-1

Foxp3 and IL-10 Expression Correlates with Parasite Burden in Lesional Tissues of Post Kala Azar Dermal Leishmaniasis (PKDL) Patients

Post kala azar dermal leishamniasis (PKDL), an unusual dermatosis develops in 5–15% of apparently cured visceral leishmaniasis cases in India and in about 60% of cases in Sudan. PKDL cases assume importance since they constitute a major human reservoir for the parasite. Inadequate treatment of VL, genetics, nutrition and immunological mechanisms that allow renewed multiplication of latent parasites or reinfection predispose to PKDL. Immunopathogenesis of PKDL is poorly understood. IL-10 is widely accepted as an immuno-suppressive cytokine and produced by diverse cell populations including, B cells, macrophages and CD4+ T cells. Natural T regulatory (nTreg) cells are subpopulation of CD4+ T cells that inhibit the response of other T cells. In this study we reported the accumulation of nTreg cells in lesion tissues of PKDL patients. Further correlation of Treg markers and IL-10 with parasite load in lesion tissues suggested a role of IL-10 and Treg in parasite establishment or persistence. Further studies are warranted to explore antigen specific IL-10 source in lesion tissues and unravel the concerted induction or accumulation of Treg in PKDL