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.
Inactivation of tumor suppressors is among the rate-limiting steps in carcinogenesis that occur during the tumor promotion stage. The translation inhibitor programmed cell death 4 (Pdcd4) suppresses ...tumorigenesis and invasion. Although Pdcd4 is not mutationally inactivated in human cancer, the mechanisms controlling Pdcd4 inactivation during tumorigenesis remain elusive. We report that tumor promoter 12-O-tetradecanoylphorbol-13-acetate exposure decreases protein levels of Pdcd4 in mouse skin papillomas and keratinocytes as well as in human HEK293 cells. This decrease is attributable to increased proteasomal degradation of Pdcd4 and is mediated by protein kinase C-dependent activation of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin-p70(S6K) and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling. Both Akt and p70(S6K) phosphorylate Pdcd4, allowing for binding of the E3-ubiquitin ligase beta-TrCP and consequently ubiquitylation. MEK-ERK signaling on the other hand facilitates the subsequent proteasomal degradation. We further show that Pdcd4 protein levels in vivo are limiting for tumor formation, establishing Pdcd4 as a haploinsufficient tumor suppressor in Pdcd4-deficient mice. Thus, because endogenous Pdcd4 levels are limiting for tumorigenesis, inhibiting signaling to Pdcd4 degradation may prove a valid strategy for cancer prevention and intervention.
Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV ...infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI.
To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells.
We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors.
IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.
While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an ...iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.
Hypoxia contributes to numerous pathophysiological conditions including inflammation-associated diseases. We characterized the impact of hypoxia on the immunometabolic cross-talk between cholesterol ...and interferon (IFN) responses. Specifically, hypoxia reduced cholesterol biosynthesis flux and provoked a compensatory activation of sterol regulatory element-binding protein 2 (SREBP2) in monocytes. Concomitantly, a broad range of interferon-stimulated genes (ISGs) increased under hypoxia in the absence of an inflammatory stimulus. While changes in cholesterol biosynthesis intermediates and SREBP2 activity did not contribute to hypoxic ISG induction, intracellular cholesterol distribution appeared critical to enhance hypoxic expression of chemokine ISGs. Importantly, hypoxia further boosted chemokine ISG expression in monocytes upon infection with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Mechanistically, hypoxia sensitized toll-like receptor 4 (TLR4) signaling to activation by SARS-CoV-2 spike protein, which emerged as a major signaling hub to enhance chemokine ISG induction following SARS-CoV-2 infection of hypoxic monocytes. These data depict a hypoxia-regulated immunometabolic mechanism with implications for the development of systemic inflammatory responses in severe cases of coronavirus disease-2019 (COVID-19).
Despite the progress to understand inflammatory reactions, mechanisms causing their resolution remain poorly understood. Prostanoids, especially prostaglandin E
(PGE
), are well-characterized ...mediators of inflammation. PGE
is produced in an inducible manner in macrophages (Mϕ) by microsomal PGE
-synthase-1 (mPGES-1), with the notion that it also conveys pro-resolving properties. We aimed to characterize the role of mPGES-1 during resolution of acute, zymosan-induced peritonitis. Experimentally, we applied the mPGES-1 inhibitor compound III (CIII) once the inflammatory response was established and confirmed its potent PGE
-blocking efficacy. mPGES-1 inhibition resulted in an incomplete removal of neutrophils and a concomitant increase in monocytes and Mϕ during the resolution process. The mRNA-seq analysis identified enhanced C-X3-C motif receptor 1 (CX3CR1) expression in resident and infiltrating Mϕ upon mPGES-1 inhibition. Besides elevated Cx3cr1 expression, its ligand CX3CL1 was enriched in the peritoneal lavage of the mice, produced by epithelial cells upon mPGES-1 inhibition. CX3CL1 not only increased adhesion and survival of Mϕ but its neutralization also completely reversed elevated inflammatory cell numbers, thereby normalizing the cellular, peritoneal composition during resolution. Our data suggest that mPGES-1-derived PGE
contributes to the resolution of inflammation by preventing CX3CL1-mediated retention of activated myeloid cells at sites of injury.
Background
Breast cancer is the leading cause of cancer‐related deaths in women, demanding new treatment options. With the advent of immune checkpoint blockade, immunotherapy emerged as a treatment ...option. In addition to lymphocytes, tumor‐associated macrophages exert a significant, albeit controversial, impact on tumor development. Pro‐inflammatory macrophages are thought to hinder, whereas anti‐inflammatory macrophages promote tumor growth. However, molecular markers to identify prognostic macrophage populations remain elusive.
Methods
We isolated two macrophage subsets, from 48 primary human breast tumors, distinguished by the expression of CD206. Their transcriptomes were analyzed via RNA‐Seq, and potential prognostic macrophage markers were validated by PhenOptics in tissue microarrays of patients with invasive breast cancer.
Results
Normal human breast tissue contained mainly CD206+ macrophages, while increased relative amounts of CD206− macrophages were observed in tumors. The presence of CD206+ macrophages correlated with a pronounced lymphocyte infiltrate and subsets of CD206+ macrophages, expressing SERPINH1 and collagen 1, or MORC4, were unexpectedly associated with improved survival of breast cancer patients. In contrast, MHCIIhi CD206− macrophages were linked with a poor survival prognosis.
Conclusion
Our data highlight the heterogeneity of tumor‐infiltrating macrophages and suggest the use of multiple phenotypic markers to predict the impact of macrophage subpopulations on cancer prognosis. We identified novel macrophage markers that correlate with the survival of patients with invasive mammary carcinoma.
Transcriptome analysis of CD206+ and CD206‐ macrophages reveals differential phenotype.
CD06+ macrophages coexpressing either MORC4 or SERPINH1 show a positive correlation with patient survival.
CD206‐ macrophages expressing MHCII are correlated with negative patient survival.
MΦ show a highly versatile phenotype depending on the receiving microenvironmental stimuli. MΦ phenotypes are grouped in three subcategories. One is classically activated MΦ (after stimulation with ...LPS or IFN-γ), and two are alternatively activated forms, known as wound-healing MΦ (induced by IL-4/IL-13) and regulatory MΦ (induced by IL-10/TGF-β). Besides cytokines, hypoxia defines MΦ functions, as shown for classically activated cells. Yet, little is known about the role of hypoxia and HIF-1 and -2 in wound-healing or regulatory MΦ. HIF target genes (such as ADM), analyzed in alternatively activated MΦ from WT and HIF-/- mice, were regulated predominantly by HIF-1 and consistently showed reduced hypoxic induction in MΦ stimulated with IL-4. To gain mechanistic insights, we analyzed HIF expression in polarized MΦ. Classically activated MΦ are characterized by the induction of HIF-1α but reduction of HIF-2α mRNA and protein, whereas wound-healing MΦ decreased HIF-1α protein expression without altering mRNA levels. Analysis of protein stability and expression after proteasomal inhibition pointed to translational regulation of HIF-1α in wound-healing MΦ. Following angiogenic-sprouting using embryonic stem cells exposed to supernatants of MΦ incubated with IL-4 under hypoxia, shorter sprouts were revealed compared with supernatants of hypoxic MΦ without IL-4. Conclusively, IL-4 reduces HIF-1α translation and thus, its activity in MΦ and concomitantly, attenuates their ability to promote angiogenesis under hypoxic conditions.
Macrophages constitute a major part of the tumor-infiltrating immune cells. Within the tumor microenvironment, they acquire an alternatively activated, tumor-supporting phenotype. Factors released by ...tumor cells are crucial for the recruitment of tumor-associated macrophages. In the present project, we aimed to understand the role of hsa-miR-200c-3p (miR-200c) in the interplay between tumor cells and macrophages. To this end, we employed a coculture system of MCF7 breast tumor cells and primary human macrophages and observed the transfer of miR-200c from apoptotic tumor cells to macrophages, which required intact CD36 receptor in macrophages. We further comprehensively determined miR-200c targets in macrophages by mRNA-sequencing and identified numerous migration-associated mRNAs to be downregulated by miR-200c. Consequently, miR-200c attenuated macrophage infiltration into 3-dimensional tumor spheroids. miR-200c-mediated reduction in infiltration further correlated with a miR-200c migration signature comprised of the four miR-200c-repressed, predicted targets PPM1F, RAB11FIB2, RDX, and MSN.
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