Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses.

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.

Enzymatic Inactivation of Oxysterols in Breast Tumor Cells Constraints Metastasis Formation by Reprogramming the Metastatic Lung Microenvironment.

Recent evidence indicates that immune cells contribute to the formation of tumor metastases by regulating the pre-metastatic niche. Whether tumor-derived factors involved in primary tumor formation play a role in metastasis formation is poorly characterized. Oxysterols act as endogenous regulators of lipid metabolism through the interaction with the nuclear Liver X Receptors-(LXR)α and LXRß. In the context of tumor development, they establish a pro-tumor environment by dampening antitumor immune responses, and by recruiting pro-angiogenic and immunosuppressive neutrophils. However, the ability of LXR/oxysterol axis to promote tumor invasion and metastasis by exploiting immune cells, is still up to debate. In this study we provide evidence that oxysterols participate in the primary growth of orthotopically implanted 4T1 breast tumors by establishing a tumor-promoting microenvironment. Furthermore, we show that oxysterols are involved in the metastatic spread of 4T1 breast tumors, since their enzymatic inactivation mediated by the sulfotransferase 2B1b, reduces the number of metastatic cells in the lungs of tumor-bearing mice. Finally, we provide evidence that oxysterols support the metastatic cascade by modifying the lung metastatic niche, particularly allowing the recruitment of tumor-promoting neutrophils. These results identify a possible new metastatic pathway to target in order to prevent metastasis formation in breast cancer patients.

The clearance of cell remnants and the regeneration of the injured muscle depend on soluble pattern recognition receptor PTX3.

OBJECTIVE: The signals causing the resolution of muscle inflammation are only partially characterized. The long pentraxin PTX3, which modulates leukocyte recruitment and activation, could contribute. METHODS: We analysed the expression of ptx3 after muscle injury and verified whether hematopoietic precursors are a source of the protein. The kinetics of regeneration and leukocytes infiltration, the accumulation of cell remnants and anti-histidyl-t-RNA synthetase autoantibodies were compared in wild-type and ptx3-deficient mice. RESULTS: Ptx3 expression was up-regulated three-five days after injury and restricted to the extracellular matrix. Cellular debris and leukocytes persisted in the muscle of ptx3-deficient mice for a long time after wild-type animals had healed. ptx3-deficient macrophages expressed receptors involved in apoptotic cell clearance and engulfed dead cells in vitro. Accumulation of cell debris in a pro-inflammatory microenvironment was not sufficient to elicit autoantibodies. CONCLUSION: PTX3 generated in response to muscle injury prompts the clearance of debris and the termination of the inflammatory response.

24-Hydroxycholesterol participates in pancreatic neuroendocrine tumor development.

Cells in the tumor microenvironment may be reprogrammed by tumor-derived metabolites. Cholesterol-oxidized products, namely oxysterols, have been shown to favor tumor growth directly by promoting tumor cell growth and indirectly by dampening antitumor immune responses. However, the cellular and molecular mechanisms governing oxysterol generation within tumor microenvironments remain elusive. We recently showed that tumor-derived oxysterols recruit neutrophils endowed with protumoral activities, such as neoangiogenesis. Here, we show that hypoxia inducible factor-1a (HIF-1α) controls the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol (24S-HC) in a pancreatic neuroendocrine tumor (pNET) model commonly used to study neoangiogenesis. The activation of the HIF-1α-24S-HC axis ultimately leads to the induction of the angiogenic switch through the positioning of proangiogenic neutrophils in proximity to Cyp46a1+ islets. Pharmacologic blockade or genetic inactivation of oxysterols controls pNET tumorigenesis by dampening the 24S-HC-neutrophil axis. Finally, we show that in some human pNET samples Cyp46a1 transcripts are overexpressed, which correlate with the HIF-1α target VEGF and with tumor diameter. This study reveals a layer in the angiogenic switch of pNETs and identifies a therapeutic target for pNET patients.

The administration of drugs inhibiting cholesterol/oxysterol synthesis is safe and increases the efficacy of immunotherapeutic regimens in tumor-bearing mice.

Tumor-derived metabolites dampen tumor-infiltrating immune cells and antitumor immune responses. Among the various metabolites produced by tumors, we recently showed that cholesterol oxidized products, namely oxysterols, favor tumor growth through the inhibition of DC migration toward lymphoid organs and by promoting the recruitment of pro-tumor neutrophils within the tumor microenvironment. Here, we tested different drugs capable of blocking cholesterol/oxysterol formation. In particular, we tested efficacy and safety of different administration schedules, and of immunotherapy-based combination of a class of compounds, namely zaragozic acids, which inhibit cholesterol pathway downstream of mevalonate formation, thus leaving intact the formation of the isoprenoids, which are required for the maturation of proteins involved in the immune cell function. We show that zaragozic acids inhibit the in vivo growth of the RMA lymphoma and the Lewis lung carcinoma (LLC) without inducing side effects. Tumor growth inhibition requires an intact immune system, as immunodeficient tumor-bearing mice do not respond to zaragozic acid treatment. Of note, the effect of zaragozic acids is accompanied by a marked reduction in the LXR target genes Abcg1, Mertk, Scd1 and Srebp-1c in the tumor microenvironment. On the other hand, zoledronate, which blocks also isoprenoid formation, did not control the LLC tumor growth. Finally, we show that zaragozic acids potentiate the antitumor effects of active and adoptive immunotherapy, significantly prolonging the overall survival of tumor-bearing mice treated with the combo zaragozic acids and TAA-loaded DCs. This study identifies zaragozic acids as new antitumor compounds exploitable for the treatment of cancer patients.

The Repair of Skeletal Muscle Requires Iron Recycling through Macrophage Ferroportin.

Macrophages recruited at the site of sterile muscle damage play an essential role in the regeneration of the tissue. In this article, we report that the selective disruption of macrophage ferroportin (Fpn) results in iron accumulation within muscle-infiltrating macrophages and jeopardizes muscle healing, prompting fat accumulation. Macrophages isolated from the tissue at early time points after injury express ferritin H, CD163, and hemeoxygenase-1, indicating that they can uptake heme and store iron. At later time points they upregulate Fpn expression, thus acquiring the ability to release the metal. Transferrin-mediated iron uptake by regenerating myofibers occurs independently of systemic iron homeostasis. The inhibition of macrophage iron export via the silencing of Fpn results in regenerating muscles with smaller myofibers and fat accumulation. These results highlight the existence of a local pathway of iron recycling that plays a nonredundant role in the myogenic differentiation of muscle precursors, limiting the adipose degeneration of the tissue.

Cholesterol metabolites and tumor microenvironment: the road towards clinical translation.

Targeting the tumor microenvironment focusing on immune cells has recently become a standard of care for some tumors. Indeed, antibodies blocking immune checkpoints (e.g., anti-CTLA-4 and anti-PD1 mAbs) have been approved by regulatory agencies for the treatment of some solid tumors based upon successes in many clinical trials. Although tumor metabolism has always attracted the attention of tumor biologists, only recently have oncologists renewed their interest in this field of tumor biology research. This has highlighted the possibility to pharmacologically target rate-limiting enzymes along key metabolic pathways of tumor cells, such as lipogenesis and aerobic glycolysis. Altered tumor metabolism has also been shown to influence the functionality of the tumor microenvironment as a whole, particularly the immune cell component of thereof. Cholesterol, oxysterols and Liver X receptors (LXRs) have been investigated in different tumor models. Recent in vitro and in vivo results point to their involvement in tumor and immune cell biology, thus making the LXR/oxysterol axis a possible target for novel antitumor strategies. Indeed, the possibility to target both tumor cell metabolism (i.e., cholesterol metabolism) and tumor-infiltrating immune cell dysfunctions induced by oxysterols might result in a synergistic antitumor effect generating long-lasting memory responses. This review will focus on the role of cholesterol metabolism with particular emphasis on the role of the LXR/oxysterol axis in the tumor microenvironment, discussing mechanisms of action, pros and cons, and strategies to develop antitumor therapies based on the modulation of this axis.

FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration.

Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2-/- γ-chain-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4+CD25+FOXP3+ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue.

Transplanted mesoangioblasts require macrophage IL-10 for survival in a mouse model of muscle injury.

The aim of this study was to verify whether macrophages influence the fate of transplanted mesoangioblasts--vessel-associated myogenic precursors--in a model of sterile toxin-induced skeletal muscle injury. We have observed that in the absence of macrophages, transplanted mesoangioblasts do not yield novel fibers. Macrophages retrieved from skeletal muscles at various times after injury display features that resemble those of immunoregulatory macrophages. Indeed, they secrete IL-10 and express CD206 and CD163 membrane receptors and high amounts of arginase I. We have reconstituted the muscle-associated macrophage population by injecting polarized macrophages before mesoangioblast injection: alternatively activated, immunoregulatory macrophages only support mesoangioblast survival and function. This action depends on the secretion of IL-10 in the tissue. Our results reveal an unanticipated role for tissue macrophages in mesoangioblast function. Consequently, the treatment of muscle disorders with mesoangioblasts should take into consideration coexisting inflammatory pathways, whose activation may prove crucial for its success.

Low hepcidin accounts for the proinflammatory status associated with iron deficiency.

Hepcidin is an antimicrobial peptide that controls systemic iron homeostasis. Hepcidin binding to its receptor ferroportin reduces iron availability, thus controlling microbial growth. In parallel it triggers an anti-inflammatory response in macrophages. Hepcidin is transcriptionally regulated by iron, through the bone morphogenetic protein-son of mothers against decapentaplegic (BMP-SMAD) pathway and by inflammation, through IL6-mediated STAT3 signaling. To investigate the mechanisms linking iron and inflammation, we treated C57BL/6 iron-deficient mice with a sublethal dose of lipopolysaccharide (LPS) and analyzed their inflammatory response in comparison with controls. We show that iron-deprived mice have a proinflammatory condition, exacerbated by LPS treatment leading to increased IL6 and TNFα mRNA in liver and spleen macrophages, and increased serum IL6 (482.29 ± 205.59 pg/mL) versus controls (69.01 ± 17.52 pg/mL; P < .05). Hepcidin was undetectable in iron-deficient mice but pretreatment with hepcidin normalized their response to LPS. Tmprss6(-/-) mice, characterized by iron deficiency and high hepcidin, show a blunted inflammatory response when challenged with LPS. Our data support a model in which the lack of hepcidin is responsible of the high inflammatory response to LPS in iron deficiency. The proinflammatory status associated with chronic iron deficiency could explain the resistance to infection seen in this condition.

High-mobility group box 1 release and redox regulation accompany regeneration and remodeling of skeletal muscle.

High-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecules, favors tissue regeneration via recruitment and activation of leukocytes and stem cells. Here we demonstrate, in a model of acute sterile muscle injury, that regeneration is accompanied by active reactive oxygen species (ROS) production counterbalanced and overcome by the generation of antioxidant moieties. Mitochondria are initially responsible for ROS formation. However, they undergo rapid disruption with almost complete disappearance. Twenty-four hours after injury, we observed a strong induction of MURF1 and atrogin-1 ubiquitin ligases, key signals in activation of the proteasome system and induction of muscle atrophy. At later time points, ROS generation is maintained by nonmitochondrial sources. The antioxidant response occurs in both regenerating fibers and leukocytes that express high levels of free thiols and antioxidant enzymes, such as superoxide dismutase 1 (SOD1) and thioredoxin. HMGB1, a protein thiol, weakly expressed in healthy muscles, increases during regeneration in parallel with the antioxidant response in both fibers and leukocytes. A reduced environment may be important to maintain HMGB1 bioactivity. Indeed, oxidation abrogates both muscle stem cell migration in response to HMGB1 and their ability to differentiate into myofibers in vitro. We propose that the early antioxidant response in regenerating muscle limits HMGB1 oxidation, thus allowing successful muscle regeneration.

Redox remodeling: a candidate regulator of HMGB1 function in injured skeletal muscle.

High-mobility group box-1 (HMGB1) is a prototypical endogenous signal that links tissue necrosis and wound healing. Extracellular HMGB1 has apparently contrasting biological actions: it sustains inflammation (with the possible establishment of autoimmunity or of self-maintaining tissue damage) while activating and recruiting stem cells, which foster tissue repair. However, little is known about the role environmental cues play in the extracellular functions of HMGB1. The skeletal muscle is an optimal tissue model to help us unravel these underlying molecular events. Here, sterile injury triggers a potent inflammatory response that includes infiltration by inflammatory leukocytes and the parallel activation, proliferation, and fusion of muscle-specific stem cells. Recent data suggest that the regulation of environmental redox is critical for the bioactivity of HMGB1, which is extremely sensitive to oxidation. Moreover, data suggest a potential role for infiltrating alternatively activated macrophages to influence the outcome of inflammatory responses to sterile skeletal muscle necrosis.

Polarization dictates iron handling by inflammatory and alternatively activated macrophages.

BACKGROUND: Macrophages play a key role in iron homeostasis. In peripheral tissues, they are known to polarize into classically activated (or M1) macrophages and alternatively activated (or M2) macrophages. Little is known on whether the polarization program influences the ability of macrophages to store or recycle iron and the molecular machinery involved in the processes. DESIGN AND METHODS: Inflammatory/M1 and alternatively activated/M2 macrophages were propagated in vitro from mouse bone-marrow precursors and polarized in the presence of recombinant interferon-γ or interleukin-4. We characterized and compared their ability to handle radioactive iron, the characteristics of the intracellular iron pools and the expression of molecules involved in internalization, storage and export of the metal. Moreover we verified the influence of iron on the relative ability of polarized macrophages to activate antigen-specific T cells. RESULTS: M1 macrophages have low iron regulatory protein 1 and 2 binding activity, express high levels of ferritin H, low levels of transferrin receptor 1 and internalize--albeit with low efficiency -iron only when its extracellular concentration is high. In contrast, M2 macrophages have high iron regulatory protein binding activity, express low levels of ferritin H and high levels of transferrin receptor 1. M2 macrophages have a larger intracellular labile iron pool, effectively take up and spontaneously release iron at low concentrations and have limited storage ability. Iron export correlates with the expression of ferroportin, which is higher in M2 macrophages. M1 and M2 cells activate antigen-specific, MHC class II-restricted T cells. In the absence of the metal, only M1 macrophages are effective. CONCLUSIONS: Cytokines that drive macrophage polarization ultimately control iron handling, leading to the differentiation of macrophages into a subset which has a relatively sealed intracellular iron content (M1) or into a subset endowed with the ability to recycle the metal (M2).

IRP1-independent alterations of cardiac iron metabolism in doxorubicin-treated mice.

Iron aggravates the cardiotoxicity of doxorubicin (DOX), a widely used anticancer anthracycline. The amount of iron in the cell is regulated by the iron regulatory proteins (IRPs)-1 and -2 that control the posttranscriptional expression of key iron metabolism genes. In vitro and cell culture studies revealed the ability of DOX to modulate the activity of both IRPs. However, conflicting data were obtained from different cell types and experimental conditions. To investigate the connection between acute DOX cardiotoxicity and the IRPs in a mammalian organism, we analyzed IRP activity and the expression of IRP target genes in the heart of mice subjected to DOX treatment. DOX exposure elicits a differential modulation of the two IRPs with reduced IRP2 activity and unchanged IRP1 activity. IRP2 downmodulation is associated with the upregulation of the ferritin L and H genes and decreased expression of the transferrin receptor 1 (TfR1). To directly test the role of IRP1 in DOX cardiotoxicity, the DOX response was analyzed in mice lacking IRP1. DOX-mediated IRP2 downmodulation and regulation of ferritin and TfR1 expression is identical in Irp1 (-/-) mice compared to wild type, as is the degree of oxidative damage of the heart assessed by thioredoxin and thiobarbituric acid reactive substance levels and by brain natriuretic peptide mRNA expression. These data demonstrate that the alterations of cardiac iron homeostasis related to acute anthracycline cardiotoxicity occur independently of IRP1. The observed IRP2 downmodulation could serve as a means to counteract DOX cardiotoxicity by reducing the "free" cellular iron pool.

Doxorubicin paradoxically protects cardiomyocytes against iron-mediated toxicity: role of reactive oxygen species and ferritin.

The cardiotoxicity induced by the anticancer anthracycline doxorubicin (DOX) is attributed to reactions between iron and reactive oxygen species (ROS) that lead to oxidative damage. We found that DOX forms ROS in H9c2 cardiomyocytes, as shown by dichlorodihydrofluorescein oxidation and the expression of stress-responsive genes such as catalase or aldose reductase. DOX also increased ferritin levels in these cells, particularly the H subunit. A considerable increase in ferritin mRNA levels showed that DOX acted at transcriptional level, but an additional potential mechanism was identified as the down-regulation of iron regulatory protein-2, post-transcriptional inhibitor of ferritin synthesis. Pretreatment with DOX protected H9c2 cells against the damage induced by subsequent exposure to ferric ammonium citrate, and experiments with (55)Fe revealed that the protection was due to the deposition of iron in ferritin. Cytoprotection was also observed when DOX was replaced by glucose/glucose oxidase, a source of H(2)O(2), thus suggesting that DOX increases ferritin synthesis through the action of ROS. This concept was supported by three more lines of evidence. (i) DOX-induced ferritin synthesis was blocked by N-acetylcysteine, a scavenger of ROS. (ii) Mitoxantrone, a ROS-forming analogue, similarly induced ferritin expression and protected the cells against iron toxicity. (iii) 5-Iminodaunorubicin, an analogue lacking ROS-forming activity, did not induce ferritin synthesis or protect the cells against iron toxicity. These results characterize a paradoxically beneficial link between anthracycline-derived ROS, increased ferritin synthesis, and resistance to iron-mediated damage. The role of iron and ROS in anthracycline-induced cardiotoxicity may, therefore, be more complex than previously believed.