Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression.

The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

Duodenal macrophages control dietary iron absorption via local degradation of transferrin.

Iron is an essential cellular metal that is important for many physiological functions including erythropoiesis and host defense. It is absorbed from the diet in the duodenum and loaded onto transferrin (Tf), the main iron transport protein. Inefficient dietary iron uptake promotes many diseases, but mechanisms regulating iron absorption remain poorly understood. By assessing mice that harbor a macrophage-specific deletion of the tuberous sclerosis complex 2 (Tsc2), a negative regulator of mechanistic target of rapamycin complex 1 (mTORC1), we found that these mice possessed various defects in iron metabolism, including defective steady-state erythropoiesis and a reduced saturation of Tf with iron. This iron deficiency phenotype was associated with an iron import block from the duodenal epithelial cells into the circulation. Activation of mTORC1 in villous duodenal CD68+ macrophages induced serine protease expression and promoted local degradation of Tf, whereas the depletion of macrophages in mice increased Tf levels. Inhibition of mTORC1 with everolimus or serine protease activity with nafamostat restored Tf levels and Tf saturation in the Tsc2-deficient mice. Physiologically, Tf levels were regulated in the duodenum during the prandial process and Citrobacter rodentium infection. These data suggest that duodenal macrophages determine iron transfer to the circulation by controlling Tf availability in the lamina propria villi.

Aberrant Lipid Metabolism in Macrophages Is Associated with Granuloma Formation in Sarcoidosis.

Rationale: Chronic sarcoidosis is a complex granulomatous disease with limited treatment options that can progress over time. Understanding the molecular pathways contributing to disease would aid in new therapeutic development. Objectives: To understand whether macrophages from patients with nonresolving chronic sarcoidosis are predisposed to macrophage aggregation and granuloma formation and whether modulation of the underlying molecular pathways influence sarcoidosis granuloma formation. Methods: Macrophages were cultivated in vitro from isolated peripheral blood CD14+ monocytes and evaluated for spontaneous aggregation. Transcriptomics analyses and phenotypic and drug inhibitory experiments were performed on these monocyte-derived macrophages. Human skin biopsies from patients with sarcoidosis and a myeloid Tsc2-specific sarcoidosis mouse model were analyzed for validatory experiments. Measurements and Main Results: Monocyte-derived macrophages from patients with chronic sarcoidosis spontaneously formed extensive granulomas in vitro compared with healthy control participants. Transcriptomic analyses separated healthy and sarcoidosis macrophages and identified an enrichment in lipid metabolic processes. In vitro patient granulomas, sarcoidosis mouse model granulomas, and those directly analyzed from lesional patient skin expressed an aberrant lipid metabolism profile and contained increased neutral lipids. Conversely, a combination of statins and cholesterol-reducing agents reduced granuloma formation both in vitro and in vivo in a sarcoidosis mouse model. Conclusions: Together, our findings show that altered lipid metabolism in sarcoidosis macrophages is associated with its predisposition to granuloma formation and suggest cholesterol-reducing therapies as a treatment option in patients.

mTOR-Mediated Regulation of Dendritic Cell Differentiation and Function.

Dendritic cells (DCs) are essential antigen-presenting cells that sample the extra- and intracellular milieu to process antigens for the instruction of T cell responses. The mammalian target of rapamycin (mTOR) network senses environmental cues and is important for numerous cellular processes. This review discusses how DCs use mTOR complexes (mTORC1 and 2) to adapt their cellular metabolism, transcriptional responses, and translation machinery to control DC development, antigen processing, cytokine production, and T cell stimulation. We present a spatiotemporal model suggesting that the mTOR network integrates pattern recognition and growth factor receptor activation with nutritional information from the cell and surrounding tissue to support T cell stimulation and tolerance. mTOR develops into a central player that regulates DC differentiation and immune functions.

MeCP2 is a naturally supercharged protein with cell membrane transduction capabilities.

The intrinsically disordered protein MeCP2 is a global transcriptional regulator encoded by the MECP2 gene. Although the structured domains of MeCP2 have been the subject of multiple studies, its unstructured regions have not been that extensively characterized. In this work, we show that MeCP2 possesses properties akin to those of supercharged proteins. By utilizing its unstructured portions, MeCP2 can successfully transduce across cell membranes and localize to heterochromatic foci in the nuclei, displaying uptake levels a third lower than a MeCP2 construct fused to the cell-penetrating peptide TAT. MeCP2 uptake can further be enhanced by the addition of compounds that promote endosomal escape following cellular trafficking by means of macropinocytosis. Using a combination of in silico prediction algorithms and live-cell imaging experiments, we mapped the sequence in MeCP2 responsible for its cellular incorporation, which bears a striking resemblance to TAT itself. Transduced MeCP2 was shown to interact with HDAC3. These findings provide valuable insight into the properties of MeCP2 and may be beneficial for devising future protein-based treatment strategies.

Interleukin-6 initiates muscle- and adipose tissue wasting in a novel C57BL/6 model of cancer-associated cachexia.

BACKGROUND: Cancer-associated cachexia (CAC) is a wasting syndrome drastically reducing efficacy of chemotherapy and life expectancy of patients. CAC affects up to 80% of cancer patients, yet the mechanisms underlying the disease are not well understood and no approved disease-specific medication exists. As a multiorgan disorder, CAC can only be studied on an organismal level. To cover the diverse aetiologies of CAC, researchers rely on the availability of a multifaceted pool of cancer models with varying degrees of cachexia symptoms. So far, no tumour model syngeneic to C57BL/6 mice exists that allows direct comparison between cachexigenic- and non-cachexigenic tumours. METHODS: MCA207 and CHX207 fibrosarcoma cells were intramuscularly implanted into male or female, 10-11-week-old C57BL/6J mice. Tumour tissues were subjected to magnetic resonance imaging, immunohistochemical-, and transcriptomic analysis. Mice were analysed for tumour growth, body weight and -composition, food- and water intake, locomotor activity, O2 consumption, CO2 production, circulating blood cells, metabolites, and tumourkines. Mice were sacrificed with same tumour weights in all groups. Adipose tissues were examined using high-resolution respirometry, lipolysis measurements in vitro and ex vivo, and radioactive tracer studies in vivo. Gene expression was determined in adipose- and muscle tissues by quantitative PCR and Western blotting analyses. Muscles and cultured myotubes were analysed histologically and by immunofluorescence microscopy for myofibre cross sectional area and myofibre diameter, respectively. Interleukin-6 (Il-6) was deleted from cancer cells using CRISPR/Cas9 mediated gene editing. RESULTS: CHX207, but not MCA207-tumour-bearing mice exhibited major clinical features of CAC, including systemic inflammation, increased plasma IL-6 concentrations (190 pg/mL, P ≤ 0.0001), increased energy expenditure (+28%, P ≤ 0.01), adipose tissue loss (-47%, P ≤ 0.0001), skeletal muscle wasting (-18%, P ≤ 0.001), and body weight reduction (-13%, P ≤ 0.01) 13 days after cancer cell inoculation. Adipose tissue loss resulted from reduced lipid uptake and -synthesis combined with increased lipolysis but was not associated with elevated beta-adrenergic signalling or adipose tissue browning. Muscle atrophy was evident by reduced myofibre cross sectional area (-21.8%, P ≤ 0.001), increased catabolic- and reduced anabolic signalling. Deletion of IL-6 from CHX207 cancer cells completely protected CHX207IL6KO -tumour-bearing mice from CAC. CONCLUSIONS: In this study, we present CHX207 fibrosarcoma cells as a novel tool to investigate the mediators and metabolic consequences of CAC in C57BL/6 mice in comparison to non-cachectic MCA207-tumour-bearing mice. IL-6 represents an essential trigger for CAC development in CHX207-tumour-bearing mice.

Metabolic support by macrophages sustains colonic epithelial homeostasis.

The intestinal epithelium has a high turnover rate and constantly renews itself through proliferation of intestinal crypt cells, which depends on insufficiently characterized signals from the microenvironment. Here, we showed that colonic macrophages were located directly adjacent to epithelial crypt cells in mice, where they metabolically supported epithelial cell proliferation in an mTORC1-dependent manner. Specifically, deletion of tuberous sclerosis complex 2 (Tsc2) in macrophages activated mTORC1 signaling that protected against colitis-induced intestinal damage and induced the synthesis of the polyamines spermidine and spermine. Epithelial cells ingested these polyamines and rewired their cellular metabolism to optimize proliferation and defense. Notably, spermine directly stimulated proliferation of colon epithelial cells and colon organoids. Genetic interference with polyamine production in macrophages altered global polyamine levels in the colon and modified epithelial cell proliferation. Our results suggest that macrophages act as "commensals" that provide metabolic support to promote efficient self-renewal of the colon epithelium.

p38 regulates the tumor suppressor PDCD4 via the TSC-mTORC1 pathway.

Programmed cell death protein 4 (PDCD4) exerts critical functions as tumor suppressor and in immune cells to regulate inflammatory processes. The phosphoinositide 3-kinase (PI3K) promotes degradation of PDCD4 via mammalian target of rapamycin complex 1 (mTORC1). However, additional pathways that may regulate PDCD4 expression are largely ill-defined. In this study, we have found that activation of the mitogen-activated protein kinase p38 promoted degradation of PDCD4 in macrophages and fibroblasts. Mechanistically, we identified a pathway from p38 and its substrate MAP kinase-activated protein kinase 2 (MK2) to the tuberous sclerosis complex (TSC) to regulate mTORC1-dependent degradation of PDCD4. Moreover, we provide evidence that TSC1 and TSC2 regulate PDCD4 expression via an additional mechanism independent of mTORC1. These novel data extend our knowledge of how PDCD4 expression is regulated by stress- and nutrient-sensing pathways.

Inactivation of mTORC2 in macrophages is a signature of colorectal cancer that promotes tumorigenesis.

The mechanistic target of rapamycin complex 2 (mTORC2) is a potentially novel and promising anticancer target due to its critical roles in proliferation, apoptosis, and metabolic reprogramming of cancer cells. However, the activity and function of mTORC2 in distinct cells within malignant tissue in vivo is insufficiently explored. Surprisingly, in primary human and mouse colorectal cancer (CRC) samples, mTORC2 signaling could not be detected in tumor cells. In contrast, only macrophages in tumor-adjacent areas showed mTORC2 activity, which was downregulated in stromal macrophages residing within human and mouse tumor tissues. Functionally, inhibition of mTORC2 by specific deletion of Rictor in macrophages stimulated tumorigenesis in a colitis-associated CRC mouse model. This phenotype was driven by a proinflammatory reprogramming of mTORC2-deficient macrophages that promoted colitis via the cytokine SPP1/osteopontin to stimulate tumor growth. In human CRC patients, high SPP1 levels and low mTORC2 activity in tumor-associated macrophages correlated with a worsened clinical prognosis. Treatment of mice with a second-generation mTOR inhibitor that inhibits mTORC2 and mTORC1 exacerbated experimental colorectal tumorigenesis in vivo. In conclusion, mTORC2 activity is confined to macrophages in CRC and limits tumorigenesis. These results suggest activation but not inhibition of mTORC2 as a therapeutic strategy for colitis-associated CRC.

Iron Regulation: Macrophages in Control.

Macrophages are sentinel cells of the innate immune system and have important functions in development, tissue homeostasis, and immunity. These phylogenetically ancient cells also developed a variety of mechanisms to control erythropoiesis and the handling of iron. Red pulp macrophages in the spleen, Kupffer cells in the liver, and central nurse macrophages in the bone marrow ensure a coordinated metabolism of iron to support erythropoiesis. Phagocytosis of senescent red blood cells by macrophages in the spleen and the liver provide a continuous delivery of recycled iron under steady-state conditions and during anemic stress. Central nurse macrophages in the bone marrow utilize this iron and provide a cellular scaffold and niche to promote differentiation of erythroblasts. This review focuses on the role of the distinct macrophage populations that contribute to efficient iron metabolism and highlight important cellular and systemic mechanisms involved in iron-regulating processes.

mTORC1 and mTORC2 as regulators of cell metabolism in immunity.

The mechanistic target of rapamycin (mTOR) pathway is an evolutionarily conserved signaling pathway that senses intra- and extracellular nutrients, growth factors, and pathogen-associated molecular patterns to regulate the function of innate and adaptive immune cell populations. In this review, we focus on the role of the mTOR complex 1 (mTORC1) and mTORC2 in the regulation of the cellular energy metabolism of these immune cells to regulate and support immune responses. In this regard, mTORC1 and mTORC2 generally promote an anabolic response by stimulating protein synthesis, glycolysis, mitochondrial functions, and lipid synthesis to influence proliferation and survival, effector and memory responses, innate training and tolerance as well as hematopoietic stem cell maintenance and differentiation. Deactivation of mTOR restores cell homeostasis after immune activation and optimizes antigen presentation and memory T-cell generation. These findings show that the mTOR pathway integrates spatiotemporal information of the environmental and cellular energy status by regulating cellular metabolic responses to guide immune cell activation. Elucidation of the metabolic control mechanisms of immune responses will help to generate a systemic understanding of the immune system.

Two different, mutually exclusively distributed, TP53 mutations in ovarian and peritoneal tumor tissues of a serous ovarian cancer patient: indicative for tumor origin?

High-grade serous ovarian cancer (HGSOC) is characterized by a TP53 mutation rate of up to 96.7% and associated with a more aggressive tumor biology. The origin of HGSOC is thought to arise either from fallopian tube secretory cells or the ovarian surface epithelium/inclusion cysts, the former with more evidence. Peritoneal tumor spread is heterogeneous, either excessive in the peritoneum (with miliary appearance) or more confined to the ovaries with only few (bigger and exophytically growing) peritoneal implants. Using RNA sequencing and DNA digital droplet polymerase chain reaction (PCR), we identified two different functional TP53 mutations in one HGSOC patient: one exclusively in the ovarian tumor mass and the other exclusively in ascites tumor cells, peritoneal tumor masses, and a lymph node metastasis. In blood, both mutations could be detected, the one from the peritoneal tumors with much higher frequency, presumably because of the higher tumor load. We conclude that this mutually exclusive distribution of two different TP53 mutations in different tumor tissues indicates the development of two independent carcinomas in the peritoneal cavity, probably one originating from a precancerous lesion in the fallopian tube and the other from the ovaries. In addition, in the patient's ascites CD45 and EpCAM, double-positive cells were found-proliferating but testing negative for the above-mentioned TP53 mutations. This mutually exclusive distribution of two TP53 mutations is probably further evidence that HGSOC can originate either from the fallopian tube or (more seldom) the ovaries, the former more prone for excessive peritoneal tumor spread.

Role of the immune system in the peritoneal tumor spread of high grade serous ovarian cancer.

The immune system plays a critical role in cancer progression and overall survival. Still, it is unclear if differences in the immune response are associated with different patterns of tumor spread apparent in high grade serous ovarian cancer patients and previously described by us. In this study we aimed to assess the role of the immune system in miliary (widespread, millet-sized lesions) and non-miliary (bigger, exophytically growing implants) tumor spread. To achieve this we comprehensively analyzed tumor tissues, blood, and ascites from 41 patients using immunofluorescence, flow cytometry, RNA sequencing, multiplexed immunoassays, and immunohistochemistry. Results showed that inflammation markers were systemically higher in miliary. In contrast, in non-miliary lymphocyte and monocyte/macrophage infiltration into the ascites was higher as well as the levels of PD-1 expression in tumor associated cytotoxic T-lymphocytes and PD-L1 expression in tumor cells. Furthermore, in ascites of miliary patients more epithelial tumor cells were present compared to non-miliary, possibly due to the active down-regulation of anti-tumor responses by B-cells and regulatory T-cells. Summarizing, adaptive immune responses prevailed in patients with non-miliary spread, whereas in patients with miliary spread a higher involvement of the innate immune system was apparent while adaptive responses were counteracted by immune suppressive cells and factors.

Small RNAs and the competing endogenous RNA network in high grade serous ovarian cancer tumor spread.

High grade serous ovarian cancer (HGSOC) is among the most deadly malignancies in women, frequently involving peritoneal tumor spread. Understanding molecular mechanisms of peritoneal metastasis is essential to develop urgently needed targeted therapies. We described two peritoneal tumor spread types in HGSOC apparent during surgery: miliary (numerous millet-sized implants) and non-miliary (few big, bulky implants). The former one is defined by a more epithelial-like tumor cell characteristic with less immune cell reactivity and with significant worse prognosis, even if corrected for typical clinicopathologic factors.23 HGSOC patients were enrolled in this study. Isolated tumor cells from fresh tumor tissues of ovarian and peritoneal origin and from ascites were used for ribosomal RNA depleted RNA and small RNA sequencing. RT-qPCR was used to validate results and an independent cohort of 32 patients to validate the impact on survival. Large and small RNA sequencing data were integrated and a new gene-miRNA set analysis method was developed.Thousands of new small RNAs (miRNAs and piwi-interacting RNAs) were predicted and a 13 small RNA signature was developed to predict spread type from formalin-fixed paraffin-embedded tissues. Furthermore, integrative analyses of RNA sequencing and small RNA sequencing data revealed a global upregulation of the competing endogenous RNA network in tumor tissues of non-miliary compared to miliary spread, i.e. higher expression of circular RNAs and long non-coding RNAs compared to coding RNAs but unchanged abundance of small RNAs. This global deregulated expression pattern could be co-responsible for the spread characteristic, miliary or non-miliary, in ovarian cancer.

Peritoneal tumor spread in serous ovarian cancer-epithelial mesenchymal status and outcome.

UNLABELLED: In this study we aimed to analyze the biological mechanisms underlying apparently different modes of peritoneal tumor spread in serous ovarian cancer: miliary (widespread, millet-like lesions) versus non-miliary (bigger, exophytically growing implants). Tumor tissues and ascites from 23 chemotherapy naive patients were analyzed by RNA-sequencing and flow cytometry. On the basis of differential gene expression between miliary and non-miliary, gene signatures were developed. A calculated tumor spread factor revealed a significant independent negative impact of miliary spread on overall survival (HR 3.77; CI95 1.14-12.39; p = 0.029) in an independent cohort of 165 serous ovarian cancer patients. Comparing previously published epithelial-mesenchymal transition (EMT) gene signatures, non-miliary spread correlated significantly with a reduced epithelial status. We conclude that serous ovarian cancer is a heterogeneous disease with distinct modes of peritoneal tumor spread, differing not only in clinical appearance, but also in molecular characteristics and outcome. EMT, peritoneal inflammation status, and therapeutic options are discussed. SIGNIFICANCE: More than half of serous epithelial ovarian cancer patients present with a newly described type of intraperitoneal tumor spread, associated with differences in the inflammation status, activated oncogenic pathways, lack of EMT, and thus reduced overall survival. Both, the diminished immune reaction and the enhanced epithelial and malignant characteristics of the tumor cells open new avenues for therapeutic options and strategies, like Catumaxomab, already in clinical use.

Correlation of circular RNA abundance with proliferation--exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues.

Circular RNAs are a recently (re-)discovered abundant RNA species with presumed function as miRNA sponges, thus part of the competing endogenous RNA network. We analysed the expression of circular and linear RNAs and proliferation in matched normal colon mucosa and tumour tissues. We predicted >1,800 circular RNAs and proved the existence of five randomly chosen examples using RT-qPCR. Interestingly, the ratio of circular to linear RNA isoforms was always lower in tumour compared to normal colon samples and even lower in colorectal cancer cell lines. Furthermore, this ratio correlated negatively with the proliferation index. The correlation of global circular RNA abundance (the circRNA index) and proliferation was validated in a non-cancerous proliferative disease, idiopathic pulmonary fibrosis, ovarian cancer cells compared to cultured normal ovarian epithelial cells, and 13 normal human tissues. We are the first to report a global reduction of circular RNA abundance in colorectal cancer cell lines and cancer compared to normal tissues and discovered a negative correlation of global circular RNA abundance and proliferation. This negative correlation seems to be a general principle in human tissues as validated with three different settings. Finally, we present a simple model how circular RNAs could accumulate in non-proliferating cells.