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BackgroundTumor progression is accompanied by dramatic remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness. The altered ECM of the tumor supports cancer growth and metastasis, but it is unknown if this effect involves modulation of T cell activity. To investigate if a high-density tumor-specific ECM could influence the ability of T cells to kill cancer cells, we here studied how T cells respond to 3D culture in different collagen densities.MethodsT cells cultured in 3D conditions surrounded by a high or low collagen density were imaged using confocal fluorescent microscopy. The effects of the different collagen densities on T cell proliferation, survival, and differentiation were examined using flow cytometry. Cancer cell proliferation in similar 3D conditions was also measured. Triple-negative breast cancer specimens were analyzed for the number of infiltrating CD8+ T cells and for the collagen density. Whole-transcriptome analyses were applied to investigate in detail the effects of collagen density on T cells. Computational analyses were used to identify transcription factors involved in the collagen density-induced gene regulation. Observed changes were confirmed by qRT-PCR analysis.ResultsT cell proliferation was significantly reduced in a high-density matrix compared to a low-density matrix and prolonged culture in a high-density matrix led to a higher ratio of CD4+ to CD8+ T cells. The proliferation of cancer cells was unaffected by the surrounding collagen-density. Consistently, we observed a reduction in the number of infiltrating CD8+ T-cells in mammary tumors with high collagen-density indicating that collagen-density has a role in regulating T cell abundance in human breast cancer.Whole-transcriptome analysis of 3D-cultured T cells revealed that a high-density matrix induces downregulation of cytotoxic activity markers and upregulation of regulatory T cell markers. These transcriptional changes were predicted to involve autocrine TGF-β signaling and they were accompanied by an impaired ability of tumor-infiltrating T cells to kill autologous cancer cells.ConclusionsOur study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0556-6) contains supplementary material, which is available to authorized users.

Long-term exposure to peroxisome proliferator-activated receptor g (PPARg) agonists such as rosiglitazone induces browning of rodent and human adipocytes; however, the transcriptional mechanisms governing this phenotypic switch in adipocytes are largely unknown. Here we show that rosiglitazone-induced browning of human adipocytes activates a comprehensive gene program that leads to increased mitochondrial oxidative capacity. Once induced, this gene program and oxidative capacity are maintained independently of rosiglitazone, suggesting that additional browning factors are activated. Browning triggers reprogramming of PPARg binding, leading to the formation of PPARg ''superenhancers'' that are selective for brown-in-white (brite) adipocytes. These are highly associated with key brite-selective genes. Based on such an association, we identified an evolutionarily conserved metabolic regulator, Kruppel-like factor 11 (KLF11), as a novel browning transcription factor in human adipocytes that is required for rosiglitazone-induced browning, including the increase in mitochondrial oxidative capacity. KLF11 is directly induced by PPARg and appears to cooperate with PPARg in a feed-forward manner to activate and maintain the brite-selective gene program.

Thyroid hormone (TH) and TH receptors (TRs) α and β act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRβ, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding-defective TRβ leads to disruption of the hypothalamic-pituitary-thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level.

Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a master regulator of adipocyte differentiation and function. We and others have previously mapped PPAR␥ binding at a genome-wide level in murine and human adipocyte cell lines and in primary human adipocytes. However, little is known about how binding patterns of PPAR␥ differ between brown and white adipocytes and among different types of white adipocytes. Here we have employed chromatin immunoprecipitation combined with deep sequencing to map and compare PPAR␥ binding in in vitro differentiated primary mouse adipocytes isolated from epididymal, inguinal, and brown adipose tissues. While these PPAR␥ binding profiles are overall similar, there are clear depot-selective binding sites. Most PPAR␥ binding sites previously mapped in 3T3-L1 adipocytes can also be detected in primary adipocytes, but there are a large number of PPAR␥ binding sites that are specific to the primary cells, and these tend to be located in closed chromatin regions in 3T3-L1 adipocytes. The depot-selective binding of PPAR␥ is associated with highly depot-specific gene expression. This indicates that PPAR␥ plays a role in the induction of genes characteristic of different adipocyte lineages and that preadipocytes from different depots are differentially preprogrammed to permit PPAR␥ lineage-specific recruitment even when differentiated in vitro.M ammals have fat depots at various locations in the body. Classically, these tissues have been characterized as either white adipose tissue (WAT) or brown adipose tissue (BAT). Both tissues store energy in the form of triglycerides; however, whereas WAT releases the energy as fatty acids that can be converted to metabolic energy in other tissues, BAT metabolizes the fatty acids in the adipocytes and releases the energy as heat. This unique energy-dispersing function of BAT relies on the high number of mitochondria in the adipocytes and the expression and activation of uncoupling protein 1 (UCP1), residing in the inner mitochondrial membrane (9). Previously, BAT was thought to be present only in newborn and hibernating mammals; however, recently it was demonstrated that also human adults have discrete BAT depots (13,38,46,58,61). It has been suggested that activation of these BAT depots could increase energy expenditure and lead to weight loss. Consistent with this, there seems to be a negative correlation between body mass index and the presence of BAT (13,68,69) Notably, it has also been indicated that different WAT depots are not identical but have different properties (57,62). It is currently unclear to what extent differences between the distinct WAT depots reflect differences at the cellular level between adipocytes in the depots or reflect context-dependent differences (e.g., microenvironment); however, recent evidence indicates that different subtypes of white adipocytes do exist. Thus, several studies have demonstrated different gene expression profiles between visceral and subcutaneous WAT and preadipocytes and adipocytes from these tiss...

Summary Physiologic turnover of interstitial collagen is mediated by a sequential pathway, in which collagen is fragmented by pericellular collagenases, endocytosed by collagen receptors, and routed to lysosomes for degradation by cathepsins. Here, we used intravital microscopy to investigate if malignant tumors, which are characterized by high rates of extracellular matrix turnover, utilize a similar collagen degradation pathway. Tumors of epithelial, mesenchymal, or neural crest origin all displayed vigorous endocytic collagen degradation. The cells engaged in this process were identified as tumor-associated macrophage (TAM)-like cells that degraded collagen in a mannose receptor-dependent manner. Accordingly, mannose receptor-deficient mice displayed increased intra-tumoral collagen. Whole transcriptome profiling uncovered a distinct extracellular matrix-catabolic signature of these collagen-degrading TAMs. Lineage-ablation studies revealed that collagen-degrading TAMs originated from circulating CCR2+ monocytes. The study identifies a function of TAMs in altering the tumor microenvironment through endocytic collagen turnover and establishes macrophages as centrally engaged in tumor-associated collagen degradation.

Epigenetic factors have been suggested to play an important role in metabolic memory by trapping and maintaining initial metabolic changes within the transcriptional regulatory machinery. In this study we fed mice a high fat diet (HFD) for seven weeks followed by additional five weeks of chow, to identify HFD-mediated changes to the hepatic transcriptional program that may persist after weight loss. Mice fed a HFD displayed increased fasting insulin levels, hepatosteatosis and major changes in hepatic gene transcription associated with modulation of H3K27Ac at enhancers, but no significant changes in chromatin accessibility, indicating that HFD-regulated gene transcription is primarily controlled by modulating the activity of pre-established enhancers. After return to the same body weight as chow fed control mice, the fasting insulin, glucose, and hepatic triglyceride levels were fully restored to normal levels. Moreover, HFD-regulated H3K27Ac and mRNA levels returned to similar levels as control mice. These data demonstrates that the transcription regulatory landscape in the liver induced by HFD is highly dynamic and can be reversed by weight loss. This provides hope for efficient treatment of early obesity-associated changes to hepatic complications by simple weight loss intervention without persistent reprograming of the liver transcriptome.

Uncoupling Proteins (UCPs) are integral ion channels residing in the inner mitochondrial membrane. UCP2 is ubiquitously expressed, while UCP3 is found primarily in muscles and adipose tissue. Although the exact molecular mechanism of action is controversial, it is generally agreed that both homologues function to facilitate mitochondrial fatty acid oxidation. UCP2 and -3 expression is activated by the peroxisome proliferatoractivated receptors (PPARs), but so far no PPAR response element has been reported in the vicinity of the Ucp2 and Ucp3 genes. Using genome-wide profiling of PPAR␥ occupancy in 3T3-L1 adipocytes we demonstrate that PPAR␥ associates with three chromosomal regions in the vicinity of the Ucp3 locus and weakly with a site in intron 1 of the Ucp2 gene. These sites are isolated from the nearest neighboring sites by >900 kb. The most prominent PPAR␥ binding site in the Ucp2 and Ucp3 loci is located in intron 1 of the Ucp3 gene and is the only site that facilitates PPAR␥ transactivation of a heterologous promoter. This site furthermore transactivates the endogenous Ucp3 promoter, and using chromatin conformation capture we show that it loops out to specifically interact with the Ucp2 promoter and intron 1. Our data indicate that PPAR␥ transactivation of both UCP2 and -3 is mediated through this novel enhancer in Ucp3 intron 1.