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Muckenthaler et al describe a novel form of hemochromatosis caused by a constitutional PIGA mutation in 3 children with associated neurologic dysfunction. Hemochromatosis results from decreased hepcidin, which is regulated by HFE, hemojuvelin (HJV), and transferrin receptor 2. HJV is a glycosylphosphatidylinositol-linked protein, so PIGA mutation leads to decreased HJV expression. Interestingly, none of the children had evidence of paroxysmal nocturnal hemoglobinuria. The cause of the novel association with central nervous system manifestations remains to be elucidated.

Anemia of inflammation (AI) is a highly prevalent comorbidity in patients affected by chronic inflammatory disorders, such as chronic kidney disease, inflammatory bowel disease, or cancer, that negatively affect disease outcome and quality of life. The pathophysiology of AI is multifactorial, with inflammatory hypoferremia and iron-restricted erythropoiesis playing a major role in the context of disease-specific factors. Here, we review the recent progress in our understanding of the molecular mechanisms contributing to iron dysregulation in AI, the impact of hypoferremia and anemia on the course of the underlying disease, and (novel) therapeutic strategies applied to treat AI.

Throughout life, macrophages are located in every tissue of the body, where their main roles are to phagocytose cellular debris and recycle aging red blood cells. In the tissue niche, they promote homeostasis through trophic, regulatory, and repair functions by responding to internal and external stimuli. This in turn polarizes macrophages into a broad spectrum of functional activation states, also reflected in their iron-regulated gene profile. The fast adaptation to the environment in which they are located helps to maintain tissue homeostasis under physiological conditions.

Twenty years ago the discovery of hepcidin deeply changed our understanding of the regulation of systemic iron homeostasis. It is now clear that hepcidin orchestrates systemic iron levels by controlling the amount of iron exported into the bloodstream through ferroportin. Hepcidin expression is increased in situations where systemic iron levels should be reduced, such as in iron overload and infection. Conversely, hepcidin is repressed during iron deficiency, hypoxia or expanded erythropoiesis, to increase systemic iron availability and sustain erythropoiesis. In this review, we will focus on molecular mechanisms of hepcidin regulation and on the pathological consequences of their disruption.

Iron is an essential co-factor for cellular processes. In the immune system, it can activate macrophages and represents a potential therapeutic for various diseases. To specifically deliver iron to macrophages, iron oxide nanoparticles are embedded in polymeric micelles of reactive polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine). Upon surface functionalization via dihydrolipoic acid, iron oxide cores act as crosslinker themselves and undergo chemoselective disulfide bond formation with the surrounding poly(S-ethylsulfonyl-l-cysteine) block, yielding glutathione-responsive core cross-linked polymeric micelles (CCPMs). When applied to primary murine and human macrophages, these nanoparticles display preferential uptake, sustained intracellular iron release, and induce a strong inflammatory response. This response is also demonstrated in vivo when nanoparticles are intratracheally administered to wild-type C57Bl/6N mice. Most importantly, the controlled release concept to deliver iron oxide in redox-responsive CCPMs induces significantly stronger macrophage activation than any other iron source at identical iron levels (e.g., Feraheme), directing to a new class of immune therapeutics.

BACKGROUND AND AIMS TGFβ/bone morphogenetic protein (BMP) signaling in the liver plays a critical role in liver disease. Growth factors, such as BMP2, BMP6, and TGFβ1, are released from LSECs and signal in a paracrine manner to hepatocytes and hepatic stellate cells to control systemic iron homeostasis and fibrotic processes, respectively. The misregulation of the TGFβ/BMP pathway affects expression of the iron-regulated hormone hepcidin, causing frequent iron overload and deficiency diseases. However, whether LSEC-secreted factors can act in an autocrine manner to maintain liver homeostasis has not been addressed so far. APPROACH AND RESULTS We analyzed publicly available RNA-sequencing data of mouse LSECs for ligand-receptor interactions and identified members of the TGFβ family (BMP2, BMP6, and TGFβ1) as ligands with the highest expression levels in LSECs that may signal in an autocrine manner. We next tested the soluble factors identified through in silico analysis in optimized murine LSEC primary cultures and mice. Exposure of murine LSEC primary cultures to these ligands shows that autocrine responses to BMP2 and BMP6 are blocked despite high expression levels of the required receptor complexes partially involving the inhibitor FK-506-binding protein 12. By contrast, LSECs respond efficiently to TGFβ1 treatment, which causes reduced expression of BMP2 through activation of activin receptor-like kinase 5. CONCLUSIONS These findings reveal that TGFβ1 signaling is functionally interlinked with BMP signaling in LSECs, suggesting druggable targets for the treatment of iron overload diseases associated with deficiency of the BMP2-regulated hormone hepcidin, such as hereditary hemochromatosis, β-thalassemia, and chronic liver diseases.

Iron is an essential micronutrient and a critical cofactor for proteins involved in fundamental processes such as oxygen transport, energy production and DNA synthesis. However, iron levels need to be tightly balanced to avoid pathological consequences of iron overload or deficiency. Genetically engineered mouse models with alterations in systemic or cellular iron handling advanced our knowledge how systemic and cellular iron homeostasis is maintained. Here, we prepared a comprehensive overview of mouse models that provide insight into mechanisms of iron regulation and/or rare or frequent iron-related disorders.

Dysregulation of cellular iron homeostasis in human breast cancer is reflected by the altered expression of regulatory proteins. The expressions of iron-related proteins in the mammary glands of cats and dogs have not been assessed. We evaluated the expressions of ferritin, ferroportin, hepcidin and transferrin receptor 1 in benign and malignant mammary gland lesions in cats and dogs. Iron deposition was detected using Perls' Prussian blue staining. We found no major differences in the expression of iron-related proteins between benign and malignant mammary gland lesions in either cats or dogs; however, these species exhibited accumulation of iron in benign lesions. Our findings provide an explanation for the absence of higher iron requirements by tumor cells in these animals. Further investigation of local iron homeostasis in cats and dogs and differences in their physiology compared to human breast cancer is required.

Summary Forkhead box P3 (FoxP3)+ regulatory T cells (Tregs) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)-2 receptor alpha chain]. Low-dose IL-2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE Treg phenotype, we studied its role through developmentally defined regulatory T cell (Treg) subsets in 45 SLE patients, 103 SLE-unaffected first-degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25-encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to Treg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4+FoxP3+CD45RO–CD31+ recent thymic emigrant Tregs. This first component effect influenced the proportions of circulating CD4+FoxP3highCD45RO+ activated Tregs. (2) In contrast, patients and unaffected relatives differed sharply in their activated Treg CD25 state: while relatives as control subjects up-regulated CD25 strongly in these cells during differentiation from naive Tregs, SLE patients specifically failed to do so. This CD25 up-regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated Tregs, but not to their circulating numbers. Both effects were found related to T cell IL-2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early Tregs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up-regulation upon peripheral Treg activation that is selectively deficient in patients. We expect that Treg-directed therapies can be monitored more effectively when taking this distinction into account.

Breast cancer epithelial cells with the CD44+/CD24-/low phenotype possess tumor-initiating cells and epithelial-mesenchymal transition (EMT) capacity. Massive parallel sequencing can be an interesting approach to deepen the molecular characterization of these cells. We characterized CD44+/CD24-/cytokeratin(Ck)+/CD45- cells isolated through flow cytometry from 43 biopsy and 6 mastectomy samples harboring different benign and malignant breast lesions. The Ion Torrent Ampliseq Cancer Hotspot panel v2 (CHPv2) was used for the identification of somatic mutations in the DNA extracted from isolated CD44+/CD24-/Ck+/CD45- cells. E-Cadherin and vimentin immunohistochemistry was performed on sections from the corresponding formalin-fixed, paraffin-embedded (FFPE) blocks. The percentage of CD44+/CD24-/Ck+/CD45- cells increased significantly from non-malignant to malignant lesions and in association with a significant increase in the expression of vimentin. Non-malignant lesions harbored only a single-nucleotide polymorphism (SNP). Mutations in the tumor suppressor p53 (TP53), NOTCH homolog 1 (NOTCH1), phosphatase and tensin homolog (PTEN), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) genes were found in isolated CD44+/CD24-/Ck+/CD45- cells from ductal carcinomas in situ (DCIS). Additional mutations in the colony-stimulating factor 1 receptor (CSF1R), ret proto-oncogene (RET), and TP53 genes were also identified in invasive ductal carcinomas (IDCs). The use of massive parallel sequencing technology for this type of application revealed to be extremely effective even when using small amounts of DNA extracted from a low number of cells. Additional studies are now required using larger cohorts to design an appropriate mutational profile for this phenotype.

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Background Iron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover. Methods We ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency. Conclusions/General significance We show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.

Macrophages release iron into the bloodstream via a membrane-bound iron export protein, ferroportin (FPN). The hepatic iron-regulatory hormone hepcidin controls FPN internalization and degradation in response to bacterial infection. Salmonella typhimurium can invade macrophages and proliferate in the Salmonella-containing vacuole (SCV). Hepcidin is reported to increase the mortality of Salmonella-infected animals by increasing the bacterial load in macrophages. Here we assess the iron levels and find that hepcidin increases iron content in the cytosol but decreases it in the SCV through FPN on the SCV membrane. Loss-of-FPN from the SCV via the action of hepcidin impairs the generation of bactericidal reactive oxygen species (ROS) as the iron content decreases. We conclude that FPN is required to provide sufficient iron to the SCV, where iron serves as a cofactor for the generation of antimicrobial ROS rather than as a nutrient for Salmonella., The effects of iron on vacuole-resident Salmonella in macrophages are unclear. Here the authors show that the bacteria are not subject to nutritional inhibition by iron deprivation, but that iron depletion in the vacuole, via the hepcidin-ferroportin axis, inhibits the bactericidal effect of oxidative burst.

Iron is an essential element and a critical component of molecules involved in energy production, cell cycle and intermediate metabolism. However, the same characteristic chemistry that makes it so biologically versatile may lead to iron-associated toxicity as a consequence of increased oxidative stress. The fact that free iron accumulates with age and generates ROS led to the hypothesis that it could be involved in the etiogenesis of several chronic diseases. Iron has been consistently linked to carcinogenesis, either through persistent failure in the redox balance or due to its critical role in cellular proliferation. Several reports have given evidence that alterations in the import, export and storage of cellular iron may contribute to breast cancer development, behavior and recurrence. In this review, we summarize the basic mechanisms of systemic and cellular iron regulation and highlight the findings that link their deregulation with breast cancer. To conclude, progresses in iron chelation therapy in breast cancer, as a tool to fight chemotherapy resistance, are also reviewed.

Disponível em: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264664/ The association of HFE (High Iron FE) major variants with breast cancer risk and behavior has been a matter of discussion for a long time. However, their impact on the expression of iron-related proteins in the breast cancer tissue has never been addressed. In the present study, hepcidin, ferroportin 1, transferrin receptor 1 (TfR1), and ferritin expressions, as well as tissue iron deposition were evaluated in a collection of samples from breast cancers patients and analyzed according to the patients’ HFE genotype. Within the group of patients with invasive carcinoma, those carrying the p.Cys282Tyr variant in heterozygosity presented a higher expression of hepcidin in lymphocytes and macrophages than wild-type or p.His63Asp carriers. An increased expression of TfR1 was also observed in all the cell types analyzed but only in p.Cys282Tyr/p.His63Asp compound heterozygous patients. A differential impact of the two HFE variants was further noticed with the observation of a significantly higher percentage of p.Cys282Tyr heterozygous patients presenting tissue iron deposition in comparison to p.His63Asp heterozygous. In the present cohort, no significant associations were found between HFE variants and classical clinicopathological markers of breast cancer behavior and prognosis. Although limited by a low sampling size, our results provide a new possible explanation for the previously reported impact of HFE major variants on breast cancer progression, i.e., not by influencing systemic iron homeostasis but rather by differentially modulating the local cellular expression of iron-related proteins and tissue iron deposition. OM is a recipient of the PhD grant SFRH/BD/2011/78184 from Fundação para a Ciência e Tecnologia (FCT). The authors also acknowledge financial support from ICBAS/AI&NSUMIB and by national funds through FCT and Ministério da Educação e Ciência (MEC) and when applicable co-funded by FEDER funds within the partnership agreement PT2020 related with the research unit number 4293. info:eu-repo/semantics/publishedVersion

BACKGROUND Cancer stem cells are tumor cells that present self-renewal, clonal tumor initiation capacity and clonal long-term repopulation potential. We have previously demonstrated that the co-expression of the breast cancer stem cell (BCSC) markers hyaluronan receptor (CD44) and aldehyde dehydrogenase-1 (ALDH1) in ductal carcinomas in situ could be determinant for disease progression. Combining these established BCSC markers with Ki-67 to evaluate quiescence we sought to identify, evaluate the distribution and estimate the mean percentages of CD44(+)ALDH1(+)Ki-67(-) breast cells. MATERIALS AND METHODS Triple-immunohistochemistry for CD44, ALDH1 and Ki-67 was applied in a series of 16 normal, 54 non-malignant and 155 malignant breast tissues. Clinical relevance was inferred by associations with markers of breast cancer behavior, progression and survival. RESULTS The mean percentages of cells with this phenotype increased significantly from non-malignant lesions to high-grade ductal carcinomas in situ, decreasing in invasive ductal carcinomas, as also evidenced by an inverse correlation with histological grade and tumor size. The mean percentage of CD44(+)ALDH1(+)Ki-67(-) cells was also significantly higher in women who developed distant metastasis and died due to breast cancer, and a significant association with human epidermal growth factor type 2 (HER2) negativity was observed. CONCLUSION Our novel findings indicate that CD44(+)ALDH1(+)Ki-67(-) tumor cells may favor distant metastasis and can predict overall survival in patients with ductal carcinomas of the breast. More importantly, quiescence may have a crucial role for tumor progression, treatment resistance and metastatic ability of BCSCs.

Breast cancer epithelial cells with the CD44

The Cancer Stem Cell (CSC) model proposes that cancer is driven by a cellular component which possesses stem cell (SC) properties, cancer stem cells (CSCs), a distinct cell-type which is tumorigenic and capable of invasion and metastasis. Enzymatic activity of aldehyde dehydrogenase-1 (ALDH1), a de-toxifying enzyme that oxidizes intracellular aldehydes, has been used as a marker of normal and malignant breast stem cells (BSCs). CD44-transmembrane protein has already been shown to possess the ability to identify breast epithelial cells with stem properties.In order to compare two of the currently most reliable BSCs markers, ALDH1 and CD44, and to correlate their expression within different breast lesions, 190 samples from breast cancer specimens were analyzed by tissue microarrays.ALDH1 expression was observed in 85.43% and CD44 in 90.3% of all samples. No overexpression was observed for ALDH1 between invasive tumors, ductal carcinomas in situ and non-malignant lesions of breast, although ALDH1 had a significant negative correlation with estrogen-receptor (ER) and progesterone-receptor (PR) status (p=0.002 and p=0.001, respectively) and a positive correlation with CD44 (p0.001). Moreover, combined overexpression of ALDH1 and CD44 was observed in ductal in situ tumors (p0.001).The combined overexpression of these markers in ductal carcinomas in situ is in agreement with the CSC model in breast cancer.

Background FOXP3+ regulatory T-cells (Tregs) in Systemic Lupus Erythematosus (SLE) are in a functionally deficient state with a characteristic reduction or absence of surface CD25 (the IL-2 receptor alpha chain). Genetic variation in the CD25-encoding IL2RA locus is associated with other autoimmune disorders. Methods We have studied Treg and Treg subset CD25 by flow cytometry and typed 24 SNPs in the IL2RA locus in 47 SLE patients, 108 SLE-unaffected first-degree relatives of SLE patients, and 61 unrelated control subjects. Results In both SLE patients and unaffected relatives, surface CD25 was found strongly reduced not only in activated, but already in circulating CD4+ FOXP3+ CD45RO-CD31 + recent thymic emigrant (RTE) Tregs. In contrast, unaffected relatives clearly differed from SLE patients in properties of activated CD4+ FOXP3highCD45RO + Tregs, which showed a CD25 upregulation versus non-activated CD45RO- Tregs in these relatives similar to control subjects, while not in SLE patients. The distinction of these two components contributing to the previously described SLE-characteristic Treg CD25 reduction was corroborated by our finding that the two components were influenced by polymorphisms in different regions of the IL2RA locus. Furthermore, we found that only RTE Treg CD25, as well as the genetic variants influencing it, were significantly related to numbers and relative frequencies of circulating activated Tregs, whereas CD25 upregulation upon Treg activation was not. Conclusions Our results point to (a) an intrathymic effect present in an extended population carrying SLE susceptibility factors that is responsible for reduced surface CD25 in early Tregs and a subsequently decreased activation capacity. This effect might be compensated in unaffected relatives by (b) CD25 upregulation upon Treg activation, which seemed functionally independent and was selectively deficient in SLE patients. This second component appears of particular interest for therapeutic targeting.

BACKGROUND: While the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored. METHODS: Immunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization. RESULTS: We confirm previous results by showing that breast cancer epithelial cells present an 'iron-utilization phenotype' with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an 'iron-donor' phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size. CONCLUSIONS: The present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context. info:eu-repo/semantics/publishedVersion