Mmp12 Is Translationally Regulated in Macrophages during the Course of Inflammation.

Despite the importance of rapid adaptive responses in the course of inflammation and the notion that post-transcriptional regulation plays an important role herein, relevant translational alterations, especially during the resolution phase, remain largely elusive. In the present study, we analyzed translational changes in inflammatory bone marrow-derived macrophages upon resolution-promoting efferocytosis. Total RNA-sequencing confirmed that apoptotic cell phagocytosis induced a pro-resolution signature in LPS/IFNγ-stimulated macrophages (Mϕ). While inflammation-dependent transcriptional changes were relatively small between efferocytic and non-efferocytic Mϕ; considerable differences were observed at the level of de novo synthesized proteins. Interestingly, translationally regulated targets in response to inflammatory stimuli were mostly downregulated, with only minimal impact of efferocytosis. Amongst these targets, pro-resolving matrix metallopeptidase 12 (Mmp12) was identified as a translationally repressed candidate during early inflammation that recovered during the resolution phase. Functionally, reduced MMP12 production enhanced matrix-dependent migration of Mϕ. Conclusively, translational control of MMP12 emerged as an efficient strategy to alter the migratory properties of Mϕ throughout the inflammatory response, enabling Mϕ migration within the early inflammatory phase while restricting migration during the resolution phase.

Functional RNA Dynamics Are Progressively Governed by RNA Destabilization during the Adaptation to Chronic Hypoxia.

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.

Apoptotic Cells induce Proliferation of Peritoneal Macrophages.

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.

The iron load of lipocalin-2 (LCN-2) defines its pro-tumour function in clear-cell renal cell carcinoma.

BACKGROUND: We aimed at clarifying the role of lipocalin-2 (LCN-2) in clear-cell renal cell carcinoma (ccRCC). Since LCN-2 was recently identified as a novel iron transporter, we explored its iron load as a decisive factor in conferring its biological function. METHODS: LCN-2 expression was analysed at the mRNA and protein level by using immunohistochemistry, RNAscope® and qRT-PCR in patients diagnosed with clear-cell renal cell carcinoma compared with adjacent healthy tissue. We measured LCN-2-bound iron by atomic absorption spectrometry from patient-derived samples and applied functional assays by using ccRCC cell lines, primary cells, and 3D tumour spheroids to verify the role of the LCN-2 iron load in tumour progression. RESULTS: LCN-2 was associated with poor patient survival and LCN-2 mRNA clustered in high- and low-expressing ccRCC patients. LCN-2 protein was found overexpressed in tumour compared with adjacent healthy tissue, whereby LCN-2 was iron loaded. In vitro, the iron load determines the biological function of LCN-2. Iron-loaded LCN-2 showed pro-tumour functions, whereas iron-free LCN-2 produced adverse effects. CONCLUSIONS: We provide new insights into the pro-tumour function of LCN-2. LCN-2 donates iron to cells to promote migration and matrix adhesion. Since the iron load of LCN-2 determines its pro-tumour characteristics, targeting either its iron load or its receptor interaction might represent new therapeutic options.

Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.

Sphingosine-1-phosphate (S1P) is involved in the regulation of important cellular processes, including immune-cell trafficking and proliferation. Altered S1P signaling is strongly associated with inflammation, cancer progression, and atherosclerosis; however, the mechanisms underlying its pathophysiologic effects are only partially understood. This study evaluated the effects of S1P in vitro and in vivo on the biosynthesis of leukotrienes (LTs), which form a class of lipid mediators involved in the pathogenesis of inflammatory diseases. Here, we report for the first time that S1P potently suppresses LT biosynthesis in Ca2+-ionophore-stimulated intact human neutrophils. S1P treatment resulted in intracellular Ca2+ mobilization, perinuclear translocation, and finally irreversible suicide inactivation of the LT biosynthesis key enzyme 5-lipoxygenase (5-LO). Agonist studies and S1P receptor mRNA expression analysis provided evidence for a S1P receptor 4-mediated effect, which was confirmed by a functional knockout of S1P4 in HL60 cells. Systemic administration of S1P in wild-type mice decreased both macrophage and neutrophil migration in the lungs in response to LPS and significantly attenuated 5-LO product formation, whereas these effects were abrogated in 5-LO or S1P4 knockout mice. In summary, targeting the 5-LO pathway is an important mechanism to explain S1P-mediated pathophysiologic effects. Furthermore, agonism at S1P4 represents a novel effective strategy in pharmacotherapy of inflammation.-Fettel, J., Kühn, B., Guillen, N. A., Sürün, D., Peters, M., Bauer, R., Angioni, C., Geisslinger, G., Schnütgen, F., Meyer zu Heringdorf, D., Werz, O., Meybohm, P., Zacharowski, K., Steinhilber, D., Roos, J., Maier, T. J. Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.

Macrophage HIF-2α regulates tumor-suppressive Spint1 in the tumor microenvironment.

In solid tumors, tumor-associated macrophages (TAMs) commonly accumulate within hypoxic areas. Adaptations to such environments evoke transcriptional changes by the hypoxia-inducible factors (HIFs). While HIF-1α is ubiquitously expressed, HIF-2α appears tissue-specific with consequences of HIF-2α expression in TAMs only being poorly characterized. An E0771 allograft breast tumor model revealed faster tumor growth in myeloid HIF-2α knockout (HIF-2αLysM-/- ) compared with wildtype (wt) mice. In an RNA-sequencing approach of FACS sorted wt and HIF-2α LysM-/- TAMs, serine protease inhibitor, Kunitz type-1 ( Spint1) emerged as a promising candidate for HIF-2α-dependent regulation. We validated reduced Spint1 messenger RNA expression and concomitant Spint1 protein secretion under hypoxia in HIF-2α-deficient bone marrow-derived macrophages (BMDMs) compared with wt BMDMs. In line with the physiological function of Spint1 as an inhibitor of hepatocyte growth factor (HGF) activation, supernatants of hypoxic HIF-2α knockout BMDMs, not containing Spint1, were able to release proliferative properties of inactive pro-HGF on breast tumor cells. In contrast, hypoxic wt BMDM supernatants containing abundant Spint1 amounts failed to do so. We propose that Spint1 contributes to the tumor-suppressive function of HIF-2α in TAMs in breast tumor development.

Lipocalin-2 and iron trafficking in the tumor microenvironment.

Iron is an essential element for virtually all organisms. It facilitates cell proliferation and growth but also contributes to major hallmarks of cancer such as tumor initiation, growth, and metastasis. Often, iron handling of tumor cells is disturbed, with altered iron acquisition, efflux, and storage. Targeting perturbed iron metabolic pathways might open opportunities towards novel approaches in cancer treatment. It is becoming clear that cells of the tumor microenvironment such as macrophages contribute to tumor progression. Since macrophages evolved a multitude of mechanisms to sequester, transport, store, and release iron it can be speculated that tumor cells educate them to supply iron to support tumor growth. Recent evidence supports the existence of transferrin-independent iron transport mechanisms in the tumor microenvironment, which points to local iron transport proteins such as lipocalin-2 and/or low molecular weight iron-trafficking substances such as siderophores. We hypothesize that tumor cells educate immune cells, i.e. macrophages in their neighborhood to make them delivering iron for the benefit of cancer progression. In particular, we pay attention to recent developments, pointing to lipocalin-2 and siderophores as alternative iron transport molecules in the tumor microenvironment.

Cholesterol metabolism in the regulation of inflammatory responses.

The importance of biologically active lipid mediators, such as prostanoids, leukotrienes, and specialized pro-resolving mediators, in the regulation of inflammation is well established. While the relevance of cholesterol in the context of atherosclerosis is also widely accepted, the role of cholesterol and its biosynthetic precursors on inflammatory processes is less comprehensively described. In the present mini-review, we summarize the current understanding of the inflammation-regulatory properties of cholesterol and relevant biosynthetic intermediates taking into account the implications of different subcellular distributions. Finally, we discuss the inflammation-regulatory effect of cholesterol homeostasis in the context of SARS-CoV-2 infections.

Hypoxia-altered cholesterol homeostasis enhances the expression of interferon-stimulated genes upon SARS-CoV-2 infections in monocytes.

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).

MicroRNA-200c Attenuates the Tumor-Infiltrating Capacity of Macrophages.

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.

Inhibition of mPGES-1 attenuates efficient resolution of acute inflammation by enhancing CX3CL1 expression.

Despite the progress to understand inflammatory reactions, mechanisms causing their resolution remain poorly understood. Prostanoids, especially prostaglandin E2 (PGE2), are well-characterized mediators of inflammation. PGE2 is produced in an inducible manner in macrophages (Mϕ) by microsomal PGE2-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 PGE2-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 PGE2 contributes to the resolution of inflammation by preventing CX3CL1-mediated retention of activated myeloid cells at sites of injury.

Import of Aspartate and Malate by DcuABC Drives H2/Fumarate Respiration to Promote Initial Salmonella Gut-Lumen Colonization in Mice.

Initial enteropathogen growth in the microbiota-colonized gut is poorly understood. Salmonella Typhimurium is metabolically adaptable and can harvest energy by anaerobic respiration using microbiota-derived hydrogen (H2) as an electron donor and fumarate as an electron acceptor. As fumarate is scarce in the gut, the source of this electron acceptor is unclear. Here, transposon sequencing analysis along the colonization trajectory of S. Typhimurium implicates the C4-dicarboxylate antiporter DcuABC in early murine gut colonization. In competitive colonization assays, DcuABC and enzymes that convert the C4-dicarboxylates aspartate and malate into fumarate (AspA, FumABC), are required for fumarate/H2-dependent initial growth. Thus, S. Typhimurium obtains fumarate by DcuABC-mediated import and conversion of L-malate and L-aspartate. Fumarate reduction yields succinate, which is exported by DcuABC in exchange for L-aspartate and L-malate. This cycle allows S. Typhimurium to harvest energy by H2/fumarate respiration in the microbiota-colonized gut. This strategy may also be relevant for commensal E. coli diminishing the S. Typhimurium infection.

Microbiota-derived hydrogen fuels Salmonella typhimurium invasion of the gut ecosystem.

The intestinal microbiota features intricate metabolic interactions involving the breakdown and reuse of host- and diet-derived nutrients. The competition for these resources can limit pathogen growth. Nevertheless, some enteropathogenic bacteria can invade this niche through mechanisms that remain largely unclear. Using a mouse model for Salmonella diarrhea and a transposon mutant screen, we discovered that initial growth of Salmonella Typhimurium (S. Tm) in the unperturbed gut is powered by S. Tm hyb hydrogenase, which facilitates consumption of hydrogen (H2), a central intermediate of microbiota metabolism. In competitive infection experiments, a hyb mutant exhibited reduced growth early in infection compared to wild-type S. Tm, but these differences were lost upon antibiotic-mediated disruption of the host microbiota. Additionally, introducing H2-consuming bacteria into the microbiota interfered with hyb-dependent S. Tm growth. Thus, H2 is an Achilles' heel of microbiota metabolism that can be subverted by pathogens and might offer opportunities to prevent infection.