HuR plays an important role in tumor cell survival mainly through posttranscriptional upregulation of prominent anti-apoptotic genes. In addition, HuR can inhibit the translation of pro-apoptotic ...factors as we could previously report for caspase-2. Here, we investigated the mechanisms of caspase-2 suppression by HuR and its contribution to chemotherapeutic drug resistance of colon carcinoma cells. In accordance with the significant drug-induced increase in cytoplasmic HuR abundance, doxorubicin and paclitaxel increased the interaction of cytoplasmic HuR with the 5'untranslated region (5'UTR) of caspase-2 as shown by RNA pull down assay. Experiments with bicistronic reporter genes furthermore indicate the presence of an internal ribosome entry site (IRES) within the caspase-2-5'UTR. Luciferase activity was suppressed either by chemotherapeutic drugs or ectopic expression of HuR. IRES-driven luciferase activity was significantly increased upon siRNA-mediated knockdown of HuR implicating an inhibitory effect of HuR on caspase-2 translation which is further reinforced by chemotherapeutic drugs. Comparison of RNA-binding affinities of recombinant HuR to two fragments of the caspase-2-5'UTR by EMSA revealed a critical HuR-binding site residing between nucleotides 111 and 241 of caspase-2-5'UTR. Mapping of critical RNA binding domains within HuR revealed that a fusion of RNA recognition motif 2 (RRM2) plus the hinge region confers a full caspase-2-5'UTR-binding. Functionally, knockdown of HuR significantly increased the sensitivity of colon cancer cells to drug-induced apoptosis. Importantly, the apoptosis sensitizing effects by HuR knockdown were rescued after silencing of caspase-2. The negative caspase-2 regulation by HuR offers a novel therapeutic target for sensitizing colon carcinoma cells to drug-induced apoptosis.
Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we ...generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
Peroxisomes are proposed to play an important role in the regulation of systemic inflammation; however, the functional role of these organelles in inflammatory responses of myeloid immune cells is ...largely unknown. In this article, we demonstrate that the nonclassical peroxisome proliferator 4-phenyl butyric acid is an efficient inducer of peroxisomes in various models of murine macrophages, such as primary alveolar and peritoneal macrophages and the macrophage cell line RAW264.7, but not in primary bone marrow-derived macrophages. Further, proliferation of peroxisomes blocked the TLR4 ligand LPS-induced proinflammatory response, as detected by the reduced induction of the proinflammatory protein cyclooxygenase (COX)-2 and the proinflammatory cytokines TNF-α, IL-6, and IL-12. In contrast, disturbing peroxisome function by knockdown of peroxisomal gene
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markedly increased the LPS-dependent upregulation of the proinflammatory proteins COX-2 and TNF-α. Specifically, induction of peroxisomes did not affect the upregulation of COX-2 at the mRNA level, but it reduced the half-life of COX-2 protein, which was restored by COX-2 enzyme inhibitors but not by proteasomal and lysosomal inhibitors. Liquid chromatography-tandem mass spectrometry analysis revealed that various anti-inflammatory lipid mediators (e.g., docosahexaenoic acid) were increased in the conditioned medium from peroxisome-induced macrophages, which blocked LPS-induced COX-2 upregulation in naive RAW264.7 cells and human primary peripheral blood-derived macrophages. Importantly, LPS itself induced peroxisomes that correlated with the regulation of COX-2 during the late phase of LPS activation in macrophages. In conclusion, our findings identify a previously unidentified role for peroxisomes in macrophage inflammatory responses and suggest that peroxisomes are involved in the physiological cessation of macrophage activation.
Interleukin-38 (IL-38) is a cytokine of the IL-1 family with a role in chronic inflammation. However, its main cellular targets and receptors remain obscure. IL-38 is highly expressed in the skin and ...downregulated in psoriasis patients. We report an investigation in cellular targets of IL-38 during the progression of imiquimod-induced psoriasis. In this model, IL-38 knockout (IL-38 KO) mice show delayed disease resolution with exacerbated IL-17-mediated inflammation, which is reversed by the administration of mature IL-38 or γδ T cell-receptor-blocking antibodies. Mechanistically, X-linked IL-1 receptor accessory protein-like 1 (IL1RAPL1) is upregulated upon γδ T cell activation to feedforward-amplify IL-17 production and is required for IL-38 to suppress γδ T cell IL-17 production. Accordingly, psoriatic IL1RAPL1 KO mice show reduced inflammation and IL-17 production by γδ T cells. Our findings indicate a role for IL-38 in the regulation of γδ T cell activation through IL1RAPL1, with consequences for auto-inflammatory disease.
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•IL-38-deficient mice display delayed resolution of imiquimod-induced psoriasis•IL-38 suppresses IL-17A production by γδ T cells•Inhibition of IL-17A production by γδ T cells requires IL1RAPL1•IL1RAPL1-deficient mice show decreased γδ T cell activation during psoriasis
Han et al. report that genetic depletion of IL-38 in mice delays the resolution of imiquimod-induced psoriasis by increasing the production of the inflammatory cytokine IL-17A by skin-infiltrating T cells. Depleting these T cells or the receptor that is targeted by IL-38 reduces psoriatic skin inflammation.
Cancer-associated fibroblasts (CAFs) in the tumor microenvironment contribute to all stages of tumorigenesis and are usually considered to be tumor-promoting cells. CAFs show a remarkable degree of ...heterogeneity, which is attributed to developmental origin or to local environmental niches, resulting in distinct CAF subsets within individual tumors. While CAF heterogeneity is frequently investigated in late-stage tumors, data on longitudinal CAF development in tumors are lacking. To this end, we used the transgenic polyoma middle T oncogene-induced mouse mammary carcinoma model and performed whole transcriptome analysis in FACS-sorted fibroblasts from early- and late-stage tumors. We observed a shift in fibroblast populations over time towards a subset previously shown to negatively correlate with patient survival, which was confirmed by multispectral immunofluorescence analysis. Moreover, we identified a transcriptomic signature distinguishing CAFs from early- and late-stage tumors. Importantly, the signature of early-stage CAFs correlated well with tumor stage and survival in human mammary carcinoma patients. A random forest analysis suggested predictive value of the complete set of differentially expressed genes between early- and late-stage CAFs on bulk tumor patient samples, supporting the clinical relevance of our findings. In conclusion, our data show transcriptome alterations in CAFs during tumorigenesis in the mammary gland, which suggest that CAFs are educated by the tumor over time to promote tumor development. Moreover, we show that murine CAF gene signatures can harbor predictive value for human cancer.
Macrophages are a highly versatile and heterogenic group of immune cells, known for their involvement in inflammatory reactions. However, our knowledge about distinct subpopulations of macrophages ...and their specific contribution to the resolution of inflammation remains incomplete. We have previously shown, in an in vivo peritonitis model, that inhibition of the synthesis of the pro-inflammatory lipid mediator prostaglandin E2 (PGE2) attenuates efficient resolution of inflammation. PGE2 levels during later stages of the inflammatory process further correlate with expression of the hyaluronan (HA) receptor Lyve1 in peritoneal macrophages. In the present study, we therefore aimed to understand if PGE2 might contribute to the regulation of Lyve1 and how this might impact inflammatory responses. In line with our in vivo findings, PGE2 synergized with dexamethasone to enhance Lyve1 expression in bone marrow-derived macrophages, while expression of the predominant hyaluronan receptor CD44 remained unaltered. PGE2-mediated Lyve1 upregulation was strictly dependent on PGE2 receptor EP2 signaling. While PGE2/dexamethasone-treated macrophages, despite their enhanced Lyve1 expression, did not show inflammatory responses upon stimulation with low (LMW) or high-molecular-weight hyaluronan (HMW)-HA, they were sensitized towards LMW-HA-dependent augmentation of lipopolysaccharide (LPS)-induced inflammatory responses. Thus, Lyve1-expressing macrophages emerged as a subpopulation of macrophages integrating inflammatory stimuli with extracellular matrix-derived signals.
Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending ...on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-β synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-β was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-β, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.
While aberrant cells are routinely recognized and removed by immune cells, tumors eventually escape innate immune responses. Infiltrating immune cells are even corrupted by the tumor to acquire a ...tumor-supporting phenotype. In line, tumor-associated macrophages are well-characterized to promote tumor progression and high levels of tumor-infiltrating macrophages are a poor prognostic marker in breast cancer. Here, we aimed to further decipher the influence of macrophages on breast tumor cells and determined global gene expression changes in three-dimensional tumor spheroids upon infiltration of macrophages. While various tumor-associated mRNAs were upregulated, expression of the cytochrome P450 family member CYP1A1 was markedly attenuated. Repression of CYP1A1 in tumor cells was elicited by a macrophage-shaped tumor microenvironment rather than by direct tumor cell-macrophage contacts. In line with changes in RNA expression profiles, macrophages enhanced proliferation of the tumor cells. Enhanced proliferation and macrophage presence further correlated with reduced CYP1A1 expression in patient tumors when compared with normal tissue. These findings are of interest in the context of combinatory therapeutic approaches involving cytotoxic and immune-modulatory compounds.
Oxygen sensing and reactivity to changes in the concentration of oxygen is a fundamental property of cell physiology. The lack of O2 (hypoxia) is transmitted into many adaptive responses, a process ...that is largely controlled by a transcription factor known as hypoxia inducible factor‐1 (HIF‐1). More recent reports suggest that besides its traditional regulation via proteasomal degradation other signaling pathways contribute to stability regulation of the HIF‐1α subunit and/or HIF‐1 transactivation. These regulatory circuits allow for the integration of HIF‐1 into scenarios of cell‐survival vs. cell‐death with the rule of the thumb that short‐term mild hypoxia maintains cell viability while prolonged and severe hypoxia provokes cell demise. Cell death pathways are associated with stabilization of the tumor suppressor p53, a response also seen under hypoxic conditions. Here we summarize recent information on accumulation of HIF‐1α and p53 under hypoxia and provide a model to explain the communication between HIF‐1 and p53 under (patho)physiological conditions.
The interaction of macrophages with apoptotic cells is required for efficient resolution of inflammation. While apoptotic cell removal prevents inflammation due to secondary necrosis, it also alters ...the macrophage phenotype to hinder further inflammatory reactions. The interaction between apoptotic cells and macrophages is often studied by chemical or biological induction of apoptosis, which may introduce artifacts by affecting the macrophages as well and/or triggering unrelated signaling pathways. Here, we set up a pure cell death system in which NIH 3T3 cells expressing dimerizable Caspase-8 were co-cultured with peritoneal macrophages in a transwell system. Phenotype changes in macrophages induced by apoptotic cells were evaluated by RNA sequencing, which revealed an unexpectedly dominant impact on macrophage proliferation. This was confirmed in functional assays with primary peritoneal macrophages and IC-21 macrophages. Moreover, inhibition of apoptosis during Zymosan-induced peritonitis in mice decreased mRNA levels of cell cycle mediators in peritoneal macrophages. Proliferation of macrophages in response to apoptotic cells may be important to increase macrophage numbers in order to allow efficient clearance and resolution of inflammation.
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