Luspatercept (RAP-536) modulates oxidative stress without affecting mutation burden in myelodysplastic syndromes.

In low-risk myelodysplastic syndrome (LR-MDS), erythropoietin (EPO) is widely used for the treatment of chronic anemia. However, initial response to EPO has time-limited effects. Luspatercept reduces red blood cell transfusion dependence in LR-MDS patients. Here, we investigated the molecular action of luspatercept (RAP-536) in an in vitro model of erythroid differentiation of MDS, and also in a in vivo PDX murine model with primary samples of MDS patients carrying or not SF3B1 mutation. In our in vitro model, RAP-536 promotes erythroid proliferation by increasing the number of cycling cells without any impact on apoptosis rates. RAP-536 promoted late erythroid precursor maturation while decreasing intracellular reactive oxygen species level. RNA sequencing of erythroid progenitors obtained under RAP-536 treatment showed an enrichment of genes implicated in positive regulation of response to oxidative stress and erythroid differentiation. In our PDX model, RAP-536 induces a higher hemoglobin level. RAP-536 did not modify variant allele frequencies in vitro and did not have any effect against leukemic burden in our PDX model. These results suggest that RAP-536 promotes in vivo and in vitro erythroid cell differentiation by decreasing ROS level without any remarkable impact on iron homeostasis and on mutated allele burden.

Heterozygous Mutations in BMP6 Pro-peptide Lead to Inappropriate Hepcidin Synthesis and Moderate Iron Overload in Humans.

BACKGROUND & AIMS: Hereditary hemochromatosis is a heterogeneous group of genetic disorders characterized by parenchymal iron overload. It is caused by defective expression of liver hepcidin, the main regulator of iron homeostasis. Iron stimulates the gene encoding hepcidin (HAMP) via the bone morphogenetic protein (BMP)6 signaling to SMAD. Although several genetic factors have been found to cause late-onset hemochromatosis, many patients have unexplained signs of iron overload. We investigated BMP6 function in these individuals. METHODS: We sequenced the BMP6 gene in 70 consecutive patients with a moderate increase in serum ferritin and liver iron levels who did not carry genetic variants associated with hemochromatosis. We searched for BMP6 mutations in relatives of 5 probands and in 200 healthy individuals (controls), as well as in 2 other independent cohorts of hyperferritinemia patients. We measured serum levels of hepcidin by liquid chromatography-tandem mass spectrometry and analyzed BMP6 in liver biopsy specimens from patients by immunohistochemistry. The functions of mutant and normal BMP6 were assessed in transfected cells using immunofluorescence, real-time quantitative polymerase chain reaction, and immunoblot analyses. RESULTS: We identified 3 heterozygous missense mutations in BMP6 (p.Pro95Ser, p.Leu96Pro, and p.Gln113Glu) in 6 unrelated patients with unexplained iron overload (9% of our cohort). These mutations were detected in less than 1% of controls. p.Leu96Pro also was found in 2 patients from the additional cohorts. Family studies indicated dominant transmission. Serum levels of hepcidin were inappropriately low in patients. A low level of BMP6, compared with controls, was found in a biopsy specimen from 1 patient. In cell lines, the mutated residues in the BMP6 propeptide resulted in defective secretion of BMP6; reduced signaling via SMAD1, SMAD5, and SMAD8; and loss of hepcidin production. CONCLUSIONS: We identified 3 heterozygous missense mutations in BMP6 in patients with unexplained iron overload. These mutations lead to loss of signaling to SMAD proteins and reduced hepcidin production. These mutations might increase susceptibility to mild-to-moderate late-onset iron overload.

Hemolytic anemia repressed hepcidin level without hepatocyte iron overload: lesson from Günther disease model.

Hemolysis occurring in hematologic diseases is often associated with an iron loading anemia. This iron overload is the result of a massive outflow of hemoglobin into the bloodstream, but the mechanism of hemoglobin handling has not been fully elucidated. Here, in a congenital erythropoietic porphyria mouse model, we evaluate the impact of hemolysis and regenerative anemia on hepcidin synthesis and iron metabolism. Hemolysis was confirmed by a complete drop in haptoglobin, hemopexin and increased plasma lactate dehydrogenase, an increased red blood cell distribution width and osmotic fragility, a reduced half-life of red blood cells, and increased expression of heme oxygenase 1. The erythropoiesis-induced Fam132b was increased, hepcidin mRNA repressed, and transepithelial iron transport in isolated duodenal loops increased. Iron was mostly accumulated in liver and spleen macrophages but transferrin saturation remained within the normal range. The expression levels of hemoglobin-haptoglobin receptor CD163 and hemopexin receptor CD91 were drastically reduced in both liver and spleen, resulting in heme- and hemoglobin-derived iron elimination in urine. In the kidney, the megalin/cubilin endocytic complex, heme oxygenase 1 and the iron exporter ferroportin were induced, which is reminiscent of significant renal handling of hemoglobin-derived iron. Our results highlight ironbound hemoglobin urinary clearance mechanism and strongly suggest that, in addition to the sequestration of iron in macrophages, kidney may play a major role in protecting hepatocytes from iron overload in chronic hemolysis.

Hepcidin as a Major Component of Renal Antibacterial Defenses against Uropathogenic Escherichia coli.

The iron-regulatory peptide hepcidin exhibits antimicrobial activity. Having previously shown hepcidin expression in the kidney, we addressed its role in urinary tract infection (UTI), which remains largely unknown. Experimental UTI was induced in wild-type (WT) and hepcidin-knockout (Hepc-/-) mice using the uropathogenic Escherichia coli CFT073 strain. Compared with infected WT mice, infected Hepc-/- mice showed a dramatic increase in renal bacterial load. Moreover, bacterial invasion was significantly dampened by the pretreatment of WT mice with hepcidin. Infected Hepc-/- mice exhibited decreased iron accumulation in the renal medulla and significant attenuation of the renal inflammatory response. Notably, we demonstrated in vitro bacteriostatic activity of hepcidin against CFT073. Furthermore, CFT073 repressed renal hepcidin, both in vivo and in cultured renal cells, and reduced phosphorylation of SMAD kinase in vivo, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. In conclusion, we provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting hepcidin offers a strategy to prevent bacterial invasion.

Antigen self-anchoring onto bacteriophage T5 capsid-like particles for vaccine design.

The promises of vaccines based on virus-like particles stimulate demand for universal non-infectious virus-like platforms that can be efficiently grafted with large antigens. Here, we harnessed the modularity and extreme affinity of the decoration protein pb10 for the capsid of bacteriophage T5. SPR experiments demonstrated that pb10 fused to mCherry or to the model antigen ovalbumin (Ova) retained picomolar affinity for DNA-free T5 capsid-like particles (T5-CLPs), while cryo-EM studies attested to the full occupancy of the 120 capsid binding sites. Mice immunization with CLP-bound pb10-Ova chimeras elicited strong long-lasting anti-Ova humoral responses involving a large panel of isotypes, as well as CD8+ T cell responses, without any extrinsic adjuvant. Therefore, T5-CLP constitutes a unique DNA-free bacteriophage capsid able to display a regular array of large antigens through highly efficient chemical-free anchoring. Its ability to elicit robust immune responses paves the way for further development of this novel vaccination platform.

Oral DAV131, a charcoal-based adsorbent, inhibits intestinal colonization by ß-lactam-resistant Klebsiella pneumoniae in cefotaxime-treated mice.

Antibiotics excreted into the intestinal tract, such as broad-spectrum cephalosporins, disrupt the indigenous microflora, affect colonization resistance (CR), and promote intestinal colonization by resistant bacteria. We tested whether oral DAV131, a charcoal-based adsorbent, would prevent colonization by a cefotaxime (CTX)-resistant Klebsiella pneumoniae strain (PUG-2) in CTX-treated mice. Mice received CTX, saline, CTX and DAV131, or saline and DAV131 for 3 days before oral challenge with 10(6) CFU of PUG-2. The fecal CTX concentrations and counts of PUG-2 were assayed. Fecal CTX disappeared when DAV131 was given concomitantly with CTX (P < 0.05), and the area under the curve of PUG-2 fecal density was significantly reduced (P < 0.01). In conclusion, reducing intestinal antibiotic exposure with DAV131 may reduce colonization by resistant strains during treatment compared to treatment with CTX only. This might open new possibilities for decreasing the impact of antibiotics on the intestinal microbiota during treatments.

Crosstalk between Acidosis and Iron Metabolism: Data from In Vivo Studies.

Iron absorption requires an acidic environment that is generated by the activity of the proton pump gastric H(+)/K(+)ATPase (ATP4), expressed in gastric parietal cells. However, hepcidin, the iron regulatory peptide that inhibits iron absorption, unexpectedly upregulates ATP4 and increases gastric acidity. Thus, a concept of link between acidosis and alterations in iron metabolism, needs to be explored. We investigated this aspect in-vivo using experimental models of NH4Cl-induced acidosis and of an iron-rich diet. Under acidosis, gastric ATP4 was augmented. Serum hepcidin was induced and its mRNA level was increased in the liver but not in the stomach, a tissue where hepcidin is also expressed. mRNA and protein levels of intestinal DMT1(Divalent Metal Transporter 1) and ferroportin were downregulated. Serum iron level and transferrin saturation remained unchanged, but serum ferritin was significantly increased. Under iron-rich diet, the protein expression of ATP4A was increased and serum, hepatic and gastric hepcidin were all induced. Taken together, these results provide evidence of in-vivo relationship between iron metabolism and acidosis. For clinical importance, we speculate that metabolic acidosis may contribute in part to the pathologic elevation of serum hepcidin levels seen in patients with chronic kidney disease. The regulation of ATP4 by iron metabolism may also be of interest for patients with hemochromatosis.

FAS-L, IL-10, and double-negative CD4- CD8- TCR alpha/beta+ T cells are reliable markers of autoimmune lymphoproliferative syndrome (ALPS) associated with FAS loss of function.

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by splenomegaly, lymphadenopathy, hypergammaglobulinemia, accumulation of double-negative TCRalphabeta(+) CD4(-)CD8(-) T cells (DNT cells), and autoimmunity. Previously, DNT cell detection and a functional defect of T cells in a FAS-induced apoptosis test in vitro had been used for ALPS diagnosis. However, a functional defect can also be detected in mutation-positive relatives (MPRs) who remain free of any ALPS-related disease. In contrast, lymphocytes from patients carrying a somatic mutation of FAS exhibit normal sensitivity to FAS-induced apoptosis in vitro. We assessed the soluble FAS-L concentration in the plasma of ALPS patients carrying FAS mutations. Overall, we showed that determination of the FAS-L represents, together with the IL-10 concentration and the DNT cell percentage, a reliable tool for the diagnosis of ALPS.