Gianfranca Corna

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).

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.

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.

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.

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

Abstract 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.

24-hydroxycholesterol participates in pancreatic neuroendocrine tumor development

Significance Oxysterols promote tumor growth directly or through the dampening of tumor-infiltrating immune cells. Whether oxysterols contribute to pancreatic neuroendocrine tumor (pNET) development and how they are generated within the pNET microenvironment are currently unknown. Here, we show that the 24S-hydroxycholesterol (24S-HC) oxysterol-generating enzyme Cyp46a1 is overexpressed during the angiogenic switch in rat insulin promoter 1–T-antigen 2 (RIP1-Tag2) pNET formation. Moreover, we report that Cyp46a1 overexpression requires hypoxia inducible factor-1a (HIF-1α). Importantly, we show that pharmacologic blockade and genetic inactivation of 24S-HC delays angiogenic switch and therefore tumor formation in RIP1-Tag2. Overexpression of Cyp46a1 transcripts in some human pNET samples suggests that targeting this axis in patients affected by pancreatic neuroendocrine tumors may be an effective therapeutic strategy. 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.

Clearance of cell remnants and regeneration of injured muscle depend on soluble pattern recognition receptor PTX3

The signals causing resolution of muscle inflammation are only partially characterized. The long pentraxin PTX3, which modulates leukocyte recruitment and activation, could contribute. We analyzed the expression of PTX3 after muscle injury and verified whether hematopoietic precursors are a source of the protein. The kinetics of regeneration and leukocyte infiltration and the accumulation of cell remnants and anti-histidyl-t-RNA synthetase autoantibodies were compared in wild-type and PTX3-deficient mice. PTX3 expression was upregulated 3 d to 5 d 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 proinflammatory microenvironment was not sufficient to elicit autoantibodies. We concluded that PTX3 generated in response to muscle injury prompts clearance of debris and termination of the inflammatory response.