Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

Protein and lipid expansion microscopy with trypsin and tyramide signal amplification for 3D imaging.

Expansion microscopy, whereby the relative positions of biomolecules are physically increased via hydrogel expansion, can be used to reveal ultrafine structures of cells under a conventional microscope. Despite its utility for achieving super-resolution imaging, expansion microscopy suffers a major drawback, namely reduced fluorescence signals caused by excessive proteolysis and swelling effects. This caveat results in a lower photon budget and disfavors fluorescence imaging over a large field of view that can cover an entire expanded cell, especially in 3D. In addition, the complex procedures and specialized reagents of expansion microscopy hinder its popularization. Here, we modify expansion microscopy by deploying trypsin digestion to reduce protein loss and tyramide signal amplification to enhance fluorescence signal for point-scanning-based imaging. We name our new methodology TT-ExM to indicate dual trypsin and tyramide treatments. TT-ExM may be applied for both antibody and lipid staining. TT-ExM displayed enhanced protein retention for endoplasmic reticulum and mitochondrial markers in COS-7 cell cultures. Importantly, TT-ExM-based lipid staining clearly revealed the complex 3D membrane structures in entire expanded cells. Through combined lipid and DNA staining, our TT-ExM methodology highlighted mitochondria by revealing their DNA and membrane structures in cytoplasm, as well as the lipid-rich structures formed via phase separation in nuclei at interphase. We also observed lipid-rich chromosome matrices in the mitotic cells. These high-quality 3D images demonstrate the practicality of TT-ExM. Thus, readily available reagents can be deployed in TT-ExM to significantly enhance fluorescence signals and generate high-quality and ultrafine-resolution images under confocal microscopy.

Autism-related KLHL17 and SYNPO act in concert to control activity-dependent dendritic spine enlargement and the spine apparatus.

Dendritic spines, the tiny and actin-rich protrusions emerging from dendrites, are the subcellular locations of excitatory synapses in the mammalian brain that control synaptic activity and plasticity. Dendritic spines contain a specialized form of endoplasmic reticulum (ER), i.e., the spine apparatus, required for local calcium signaling and that is involved in regulating dendritic spine enlargement and synaptic plasticity. Many autism-linked genes have been shown to play critical roles in synaptic formation and plasticity. Among them, KLHL17 is known to control dendritic spine enlargement during development. As a brain-specific disease-associated gene, KLHL17 is expected to play a critical role in the brain, but it has not yet been well characterized. In this study, we report that KLHL17 expression in mice is strongly regulated by neuronal activity and KLHL17 modulates the synaptic distribution of synaptopodin (SYNPO), a marker of the spine apparatus. Both KLHL17 and SYNPO are F-actin-binding proteins linked to autism. SYNPO is known to maintain the structure of the spine apparatus in mature spines and contributes to synaptic plasticity. Our super-resolution imaging using expansion microscopy demonstrates that SYNPO is indeed embedded into the ER network of dendritic spines and that KLHL17 is closely adjacent to the ER/SYNPO complex. Using mouse genetic models, we further show that Klhl17 haploinsufficiency and knockout result in fewer dendritic spines containing ER clusters and an alteration of calcium events at dendritic spines. Accordingly, activity-dependent dendritic spine enlargement and neuronal activation (reflected by extracellular signal-regulated kinase (ERK) phosphorylation and C-FOS expression) are impaired. In addition, we show that the effect of disrupting the KLHL17 and SYNPO association is similar to the results of Klhl17 haploinsufficiency and knockout, further strengthening the evidence that KLHL17 and SYNPO act together to regulate synaptic plasticity. In conclusion, our findings unravel a role for KLHL17 in controlling synaptic plasticity via its regulation of SYNPO and synaptic ER clustering and imply that impaired synaptic plasticity contributes to the etiology of KLHL17-related disorders.

Hepatic Fis1 regulates mitochondrial integrated stress response and improves metabolic homeostasis.

Mitophagy and mitochondrial integrated stress response (ISR) are 2 primary protective mechanisms to maintain functional mitochondria. Whether these 2 processes are coordinately regulated remains unclear. Here we show that mitochondrial fission 1 protein (Fis1), which is required for completion of mitophagy, serves as a signaling hub linking mitophagy and ISR. In mouse hepatocytes, high fat diet (HFD) feeding induces unresolved oxidative stress, defective mitophagy and enhanced type I interferon (IFN-I) response implicated in promoting metabolic inflammation. Adenoviral-mediated acute hepatic Fis1 overexpression is sufficient to reduce oxidative damage and improve glucose homeostasis in HFD-fed mice. RNA-Seq analysis reveals that Fis1 triggers a retrograde mitochondria-to-nucleus communication upregulating ISR genes encoding anti-oxidant defense, redox homeostasis, and proteostasis pathways. Fis1-mediated ISR also suppresses expression of IFN-I-stimulated genes through activating transcription factor 5 (Atf5), which inhibits the transactivation activity of interferon regulatory factor 3 (Irf3) known to control IFN-I production. Metabolite analysis demonstrates that Fis1 activation leads to accumulation of fumarate, a TCA cycle intermediate capable of increasing Atf5 activity. Consequently, hepatic Atf5 overexpression or monomethyl fumarate (MMF) treatment improves glucose homeostasis in HFD-fed mice. Collectively, these results support the potential use of small molecules targeting the Fis1-Atf5 axis, such as MMF, to treat metabolic diseases.

Negative Regulation of the Differentiation of Flk2- CD34- LSK Hematopoietic Stem Cells by EKLF/KLF1.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and Eklf gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2- CD34- Lin- Sca1+ c-Kit+ (LSK)-HSC. In an interesting correlation, Eklf is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor ß subunit (CSF2RB). As a result, Flk2- CD34- LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2- CD34- LSK-HSCs.

Bmal1 integrates mitochondrial metabolism and macrophage activation.

Metabolic pathways and inflammatory processes are under circadian regulation. Rhythmic immune cell recruitment is known to impact infection outcomes, but whether the circadian clock modulates immunometabolism remains unclear. We find that the molecular clock Bmal1 is induced by inflammatory stimulants, including Ifn-γ/lipopolysaccharide (M1) and tumor-conditioned medium, to maintain mitochondrial metabolism under metabolically stressed conditions in mouse macrophages. Upon M1 stimulation, myeloid-specific Bmal1 knockout (M-BKO) renders macrophages unable to sustain mitochondrial function, enhancing succinate dehydrogenase (SDH)-mediated mitochondrial production of reactive oxygen species as well as Hif-1α-dependent metabolic reprogramming and inflammatory damage. In tumor-associated macrophages, aberrant Hif-1α activation and metabolic dysregulation by M-BKO contribute to an immunosuppressive tumor microenvironment. Consequently, M-BKO increases melanoma tumor burden, whereas administering the SDH inhibitor dimethyl malonate suppresses tumor growth. Therefore, Bmal1 functions as a metabolic checkpoint that integrates macrophage mitochondrial metabolism, redox homeostasis and effector functions. This Bmal1-Hif-1α regulatory loop may provide therapeutic opportunities for inflammatory diseases and immunotherapy.

Interleukin-13 drives metabolic conditioning of muscle to endurance exercise.

Repeated bouts of exercise condition muscle mitochondria to meet increased energy demand-an adaptive response associated with improved metabolic fitness. We found that the type 2 cytokine interleukin-13 (IL-13) is induced in exercising muscle, where it orchestrates metabolic reprogramming that preserves glycogen in favor of fatty acid oxidation and mitochondrial respiration. Exercise training-mediated mitochondrial biogenesis, running endurance, and beneficial glycemic effects were lost in Il13-/- mice. By contrast, enhanced muscle IL-13 signaling was sufficient to increase running distance, glucose tolerance, and mitochondrial activity similar to the effects of exercise training. In muscle, IL-13 acts through both its receptor IL-13Rα1 and the transcription factor Stat3. The genetic ablation of either of these downstream effectors reduced running capacity in mice. Thus, coordinated immunological and physiological responses mediate exercise-elicited metabolic adaptations that maximize muscle fuel economy.

FAM13A Represses AMPK Activity and Regulates Hepatic Glucose and Lipid Metabolism.

Obesity commonly co-exists with fatty liver disease with increasing health burden worldwide. Family with Sequence Similarity 13, Member A (FAM13A) has been associated with lipid levels and fat mass by genome-wide association studies (GWAS). However, the function of FAM13A in maintaining metabolic homeostasis in vivo remains unclear. Here, we demonstrated that rs2276936 in this locus has allelic-enhancer activity in massively parallel reporter assays (MPRA) and reporter assay. The DNA region containing rs2276936 regulates expression of endogenous FAM13A in HepG2 cells. In vivo, Fam13a-/- mice are protected from high-fat diet (HFD)-induced fatty liver accompanied by increased insulin sensitivity and reduced glucose production in liver. Mechanistically, loss of Fam13a led to the activation of AMP-activated protein kinase (AMPK) and increased mitochondrial respiration in primary hepatocytes. These findings demonstrate that FAM13A mediates obesity-related dysregulation of lipid and glucose homeostasis. Targeting FAM13A might be a promising treatment of obesity and fatty liver disease.

Macrophage alternative activation confers protection against lipotoxicity-induced cell death.

OBJECTIVE: Alternative activation (M2) of adipose tissue resident macrophage (ATM) inhibits obesity-induced metabolic inflammation. The underlying mechanisms remain unclear. Recent studies have shown that dysregulated lipid homeostasis caused by increased lipolysis in white adipose tissue (WAT) in the obese state is a trigger of inflammatory responses. We investigated the role of M2 macrophages in lipotoxicity-induced inflammation. METHODS: We used microarray experiments to profile macrophage gene expression regulated by two M2 inducers, interleukin-4 (Il-4), and peroxisome proliferator-activated receptor delta/gamma (Pparδ/Pparγ) agonists. Functional validation studies were performed in bone marrow-derived macrophages and mice deprived of the signal transducer and activator of transcription 6 gene (Stat6; downstream effector of Il-4) or Pparδ/Pparγ genes (downstream effectors of Stat6). Palmitic acid (PA) and ß-adrenergic agonist were employed to induce macrophage lipid loading in vitro and in vivo, respectively. RESULTS: Profiling of genes regulated by Il-4 or Pparδ/Pparγ agonists reveals that alternative activation promotes the cell survival program, while inhibiting that of inflammation-related cell death. Deletion of Stat6 or Pparδ/Pparγ increases the susceptibility of macrophages to PA-induced cell death. NLR family pyrin domain containing 3 (Nlrp3) inflammasome activation by PA in the presence of lipopolysaccharide is also increased in Stat6-/- macrophages and to a lesser extent, in Pparδ/γ-/- macrophages. In concert, ß-adrenergic agonist-induced lipolysis results in higher levels of cell death and inflammatory markers in ATMs derived from myeloid-specific Pparδ/γ-/- or Stat6-/- mice. CONCLUSIONS: Our data suggest that ATM cell death is closely linked to metabolic inflammation. Within WAT where concentrations of free fatty acids fluctuate, M2 polarization regulated by the Stat6-Ppar axis enhances ATM's tolerance to lipid-mediated stress, thereby maintaining the homeostatic state.

Skeletal muscle interleukin 15 promotes CD8(+) T-cell function and autoimmune myositis.

BACKGROUND: Interleukin 15 (IL-15) is thought to be abundant in the skeletal muscle under steady state conditions based on RNA expression; however, the IL-15 RNA level may not reflect the protein level due to post-transcriptional regulation. Although exogenous protein treatment and overexpression studies indicated IL-15 functions in the skeletal muscle, how the skeletal muscle cell uses IL-15 remains unclear. In myositis patients, IL-15 protein is up-regulated in the skeletal muscle. Given the supporting role of IL-15 in CD8(+) T-cell survival and activation and the pathogenic role of cytotoxic CD8(+) T cells in polymyositis and inclusion-body myositis, we hypothesize that IL-15 produced by the inflamed skeletal muscle promotes myositis via CD8(+) T cells. METHODS: Expression of IL-15 and IL-15 receptors at the protein level by skeletal muscle cells were examined under steady state and cytokine stimulation conditions. The functions of IL-15 in the skeletal muscle were investigated using Il15 knockout (Il15 (-/-) ) mice. The immune regulatory role of skeletal muscle IL-15 was determined by co-culturing cytokine-stimulated muscle cells and memory-like CD8(+) T cells in vitro and by inducing autoimmune myositis in skeletal-muscle-specific Il15 (-/-) mice. RESULTS: We found that the IL-15 protein was not expressed by skeletal muscle cells under steady state condition but induced by tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) stimulation and expressed as IL-15/IL-15 receptor alpha (IL-15Rα) complex. Skeletal muscle cells expressed a scanty amount of IL-15 receptor beta (IL-15Rß) under either conditions and only responded to a high concentration of IL-15 hyperagonist, but not IL-15. Consistently, deficiency of endogenous IL-15 affected neither skeletal muscle growth nor its responses to TNF-α and IFN-γ. On the other hand, the cytokine-stimulated skeletal muscle cells presented antigen and provided IL-15 to promote the effector function of memory-like CD8(+) T cells. Genetic ablation of Il15 in skeletal muscle cells greatly ameliorated autoimmune myositis in mice. CONCLUSIONS: These findings together indicate that skeletal muscle IL-15 directly regulates immune effector cells but not muscle cells and thus presents a potential therapeutic target for myositis.

The interleukin-15 system suppresses T cell-mediated autoimmunity by regulating negative selection and nT(H)17 cell homeostasis in the thymus.

The interleukin-15 (IL-15) system is important for regulating both innate and adaptive immune responses, however, its role in autoimmune disease remained unclear. Here we found that Il15(-/-) and Il15ra(-/-) mice spontaneously developed late-onset autoimmune phenotypes. CD4(+) T cells of the knockout mice showed elevated autoreactivity as demonstrated by the induction of lymphocyte infiltration in the lacrimal and salivary glands when transferred into nude mice. The antigen-presenting cells in the thymic medullary regions expressed IL-15 and IL-15Rα, whose deficiency resulted in insufficient negative selection and elevated number of natural IL-17A-producing CD4(+) thymocytes. These findings reveal previously unknown functions of the IL-15 system in thymocyte development, and thus a new layer of regulation in T cell-mediated autoimmunity.

Adipocyte IL-15 regulates local and systemic NK cell development.

NK cell development and homeostasis require IL-15 produced by both hematopoietic and parenchymal cells. Certain hematopoietic IL-15 sources, such as macrophages and dendritic cells, are known, whereas the source of parenchymal IL-15 remains elusive. Using two types of adipocyte-specific Il15(-/-) mice, we identified adipocytes as a parenchymal IL-15 source that supported NK cell development nonredundantly. Both adipocyte-specific Il15(-/-) mice showed reduced IL-15 production specifically in the adipose tissue but impaired NK cell development in the spleen and liver in addition to the adipose tissue. We also found that the adipose tissue harbored NK progenitors as other niches (e.g. spleen) for NK cell development, and that NK cells derived from transplanted adipose tissue populated the recipient's spleen and liver. These findings suggest that adipocyte IL-15 contributes to systemic NK cell development by supporting NK cell development in the adipose tissue, which serves as a source of NK cells for other organs.

Different NK cell developmental events require different levels of IL-15 trans-presentation.

NK cell development requires IL-15, which is "trans-presented" to IL-15Rßγ on NK cells by IL-15Rα on other cells. In this study, we report that different levels of IL-15 trans-presentation are required for different NK cell developmental events to reach full maturation status. Because the IL-15Rα intracellular domain has the capacity to recruit signaling molecules, we generated knockin and transgenic (Tg) mice that lack the intracellular domain to assess the role of the IL-15 trans-presentation level independent of the function of this domain. The level of IL-15Rα on various cells of these mice follows the order WT > Tg6 > knockin > Tg1 ≥ knockout. Bone marrow (BM)-derived dendritic cells prepared from these mice induced Stat5 phosphorylation in NK cells. The level of phospho-Stat5 correlated with the level of IL-15Rα on BMDCs, thus offering the opportunity to study quantitative effects of IL-15 trans-presentation on NK cell development in vivo. We found that NK cell homeostasis, mature NK cell differentiation, and acquisition of Ly49 receptor and effector functions require different levels of IL-15 trans-presentation input to achieve full status. All NK cell developmental events examined were quantitatively regulated by the IL-15Rα level of BM-derived and radiation-resistant accessory cells, but not by IL-15Rα of NK cells. We also found that IL-15Rα of radiation-resistant cells was more potent than IL-15Rα of BM-derived accessory cells in support of stage 2 to stage 3 splenic mNK differentiation. In summary, each examined developmental event required a particular level of IL-15 trans-presentation by accessory cells.

IL-15 does not affect IEL development in the thymus but regulates homeostasis of putative precursors and mature CD8 alpha alpha+ IELs in the intestine.

Mice devoid of the IL-15 system lose over 90% of CD8alphaalpha(+) TCRalphabeta and TCRgammadelta intestinal intraepithelial lymphocytes (iIELs). Previous work revealed that IL-15Ralpha and IL-15 expressed by parenchymal cells, but not by bone marrow-derived cells, are required for normal CD8alphaalpha(+) iIEL homeostasis. However, it remains unclear when and how the IL-15 system affects CD8alphaalpha(+) iIELs through their development. This study found that IL-15Ralpha is dispensable for the thymic stage of CD8alphaalpha(+) TCRalphabeta and TCRgammadelta iIEL development but is required for the maintenance and/or differentiation of the putative lineage marker negative precursors in the intestinal epithelium, especially for the most mature CD8 single positive subset. Moreover, the IL-15 system directly supports the survival of mature CD8alphaalpha(+) iIEL in vivo. Taken together, this study suggests that regulation of CD8alphaalpha(+) iIEL homeostasis by the IL-15 system does not occur in the thymus but involves mature cells and putative precursors in the intestine.

IL-15Ralpha is a negative regulator of TCR-activated proliferation in CD4+ T cells.

Although IL-15 is known to be a T cell growth factor, the function in T cells of IL-15Ralpha, its high affinity receptor, remains unclear. We found that murine IL-15Ralpha(-/-) CD4(+) T cells hyperproliferated in response to TCR stimulation, in vitro and in vivo, and displayed a lower TCR activation threshold than wild-type CD4(+) T cells. TCR-induced activation of Zap70 and of the phospholipase C-gamma1-NFATp, Ras-ERK-c-Fos, and Rac-JNK-c-Jun pathways was all augmented in IL-15Ralpha(-/-) CD4(+) T cells. This in turn led to earlier IL-2Ralpha induction and higher IL-2 production, which most likely contribute to the hyperproliferation of IL-15Ralpha(-/-) CD4(+) T cells. Exogenous IL-15 reduced levels of TCR-activated signals, transcription factors, IL-2, and IL-2Ralpha, and division in wild-type CD4(+) T cells. These results reveal IL-15Ralpha to be a negative regulator for CD4(+) T cell activation and demonstrate a novel layer of regulation of TCR signaling by a cytokine system.