Pro-Resolving Lipid Mediators (SPMs) and Their Actions in Regulating miRNA in Novel Resolution Circuits in Inflammation

Unresolved inflammation is associated with several widely occurring diseases such as arthritis, periodontal diseases, cancer, and atherosclerosis. Endogenous mechanisms that curtail excessive inflammation and prompt its timely resolution are of considerable interest. In recent years, previously unrecognized chemical mediators derived from polyunsaturated fatty acids were identified that control the acute inflammatory response by activating local resolution programs. Among these are the so-called specialized pro-resolving lipid mediators (SPMs) that include lipoxins (LX), resolvins (Rv), protectins (PD), and maresins (MaR), because they are enzymatically biosynthesized during resolution of self-limited inflammation. They each possess distinct chemical structures and regulate cellular pathways by their ability to activate pro-resolving G-protein coupled receptors (GPCRs) in a stereospecific manner. For instance, RvD1 controls several miRNAs of interest in self-limited acute inflammation that counter-regulate the mediators and proteins that are involved in inflammation. Here, we overview some of the biosynthesis and mechanisms of SPM actions with focus on the recently reported miR involved in their pro-resolving responses that underscore their beneficial actions in the regulation of acute inflammation and its timely resolution. The elucidation of these mechanisms operating in vivo to keep acute inflammation within physiologic boundaries as well as stimulate resolution have opened resolution pharmacology and many new opportunities to target inflammation-related human pathologies via activating resolution mechanisms

Mechanisms of endothelial cell dysfunction in cystic fibrosis

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans‑endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a β2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF

Lipoxin Receptors

Lipoxins (LXs) represent a class of arachidonic acid (AA) metabolites that carry potent immunoregulatory and anti-inflammatory properties, LXA4 and LXB4 being the main components of this series. LXs are generated by cooperation between 5-lipoxygenase (LO) and 12- or 15-LO during cell-cell interactions or by single cell types. LX epimers at carbon 15, the 15-epi-LXs, are formed by aspirin-acetylated cyclooxygenase-2 (COX-2) in cooperation with 5-LO. 15-epi-LXA4 is also termed aspirin-triggered LX (ATL). In vivo studies with stable LX and ATL analogs have established that these eicosanoids possess potent anti-inflammatory activities. A LXA4 receptor has been cloned. It belongs to the family of chemotactic receptors and clusters with formyl peptide receptors on chromosome 19. Therefore, it was initially denominated formyl peptide receptor like 1 (FPRL1). This receptor binds with high affinity and stereoselectivity LXA4 and ATL. It also recognizes a variety of peptides, synthetic, endogenously generated, or disease associated, but with lower affinity compared to LXA4. For this reason, this receptor has been renamed ALX. This review summarizes the current knowledge on ALX expression, signaling, and potential pathophysiological role. The involvement of additional recognition sites in LX bioactions is also discussed

Roles, Actions, and Therapeutic Potential of Specialized Pro-resolving Lipid Mediators for the Treatment of Inflammation in Cystic Fibrosis

Non-resolving inflammation is the main mechanism of morbidity and mortality among patients suffering from cystic fibrosis (CF), the most common life-threatening human genetic disease. Resolution of inflammation is an active process timely controlled by endogenous specialized pro-resolving lipid mediators (SPMs) produced locally in inflammatory loci to restrain this innate response, prevent further damages to the host, and permit return to homeostasis. Lipoxins, resolvins, protectins, and maresins are SPM derived from polyunsaturated fatty acids that limit excessive leukocyte infiltration and pro-inflammatory signals, stimulate innate microbial killing, and enhance resolution. Their unique chemical structures, receptors, and bioactions are being elucidated. Accruing data indicate that SPMs carry protective functions against unrelenting inflammation and infections in preclinical models and human CF systems. Here, we reviewed their roles and actions in controlling resolution of inflammation, evidence for their impairment in CF, and proofs of principle for their exploitation as innovative, non-immunosuppressive drugs to address inflammation and infections in CF

Resolution of inflammation: a new therapeutic frontier

Dysregulated inflammation is a central pathological process in diverse disease states. Traditionally, therapeutic approaches have sought to modulate the pro- or anti-inflammatory limbs of inflammation, with mixed success. However, insight into the pathways by which inflammation is resolved has highlighted novel opportunities to pharmacologically manipulate these processes — a strategy that might represent a complementary (and perhaps even superior) therapeutic approach. This Review discusses the state of the art in the biology of resolution of inflammation, highlighting the opportunities and challenges for translational research in this field

Video-Making and Knowledge Production in the Classroom

This dialogue is co-written by the instructor and six students in an undergraduate course on global feminisms at the Pennsylvania State University. We focus on a course assignment where students made a short video to debunk widely held societal misconceptions about women’s roles and gendered expectations regarding health, development, and environmental change across multiple global locations. We reflect upon the process of knowledge production, video as a medium of knowledge dissemination, and student efforts to assess videos made by their peers. We consider strategies that worked and others that could be improved for designing a similar assignment. Through our collective dialogue, we suggest that videos provide a fruitful entry point for discussions about knowledge making how information and misinformation circulates in this medium, and topically, focusing on countering misinformation using scholarly sources. Further, the process of co-writing this piece offered additional opportunities for considering reflexive knowledge-making practices in the classroom, and the significance of instructor-student partnerships in designing more meaningful assignments

Impaired pro‐resolving mechanisms promote abnormal NETosis , fueling autoimmunity in sickle cell disease

Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorders with still high mortality and morbidity and limited therapeutic options. SCD is characterized by anemia, chronic hemolysis, and acute vaso-occlusive painful crises. The biocomplexity of SCD goes beyond red cells, involving neutrophils and soluble factors such as cytokines or alternative complement pathway intensively cross-talking with vascular endothelial cells. In addition, in SCD, the overactivation of neutrophils contributes to the production of neutrophil extracellular traps (NETs) (1, 2). This might trigger endothelial vascular injury, promoting acute sickle cell related events and increasing the risk of infections in patients with SC

Resolvin D1 improves airway inflammation and exercise capacity in cystic fibrosis lung disease

Mucus plugging and non-resolving inflammation are inherent features of cystic fibrosis (CF) that may lead to progressive lung disease and exercise intolerance, which are the main causes of morbidity and mortality for people with CF. Therefore, understanding the influence of mucus on basic mechanisms underlying the inflammatory response and identifying strategies to resolve mucus-driven airway inflammation and consequent morbidity in CF are of wide interest. Here, we investigated the effects of the proresolving lipid mediator resolvin (Rv) D1 on mucus-related inflammation as a proof-of-concept to alleviate the burden of lung disease and restore exercise intolerance in CF. We tested the effects of RvD1 on inflammatory responses of human organotypic airways and leukocytes to CF mucus and of humanized mice expressing the epithelial Na + channel (βENaC-Tg) having CF-like mucus obstruction, lung disease, and physical exercise intolerance. RvD1 reduced pathogenic phenotypes of CF-airway supernatant (ASN)-stimulated human neutrophils, including loss of L-selectin shedding and CD16. RNASeq analysis identified select transcripts and pathways regulated by RvD1 in ASN-stimulated CF bronchial epithelial cells that are involved in sugar metabolism, NF-κB activation and inflammation, and response to stress. In in vivo inflammation using βENaC TG mice, RvD1 reduced total leukocytes, PMN, and interstitial Siglec-MΦ when given at 6–8 weeks of age, and in older mice at 10–12 weeks of age, along with the decrease of pro-inflammatory chemokines and increase of anti-inflammatory IL-10. Furthermore, RvD1 treatment promoted the resolution of pulmonary exacerbation caused by Pseudomonas aeruginosa infection and significantly enhanced physical activity and energy expenditure associated with mucus obstruction, which was impaired in βENaC-Tg mice compared with wild-type. These results demonstrate that RvD1 can rectify features of CF and offer proof-of-concept for its therapeutic application in this and other muco-obstructive lung diseases. © 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in β-thalassemic mice

Mitapivat, a pyruvate kinase activator, improves transfusion burden and reduces iron overload in β-thalassemic mice β-thalassemia (β-thal) is a genetic red cell disorder characterized by chronic hemolytic anemia due to ineffective erythropoiesis and reduced red cell survival.1-3 Chronic transfusion and intensive iron chelation are standard treatments for β-thalassemic syndromes,1 but new therapeutic options are being developed, including gene therapy4 and novel pharmacologic approaches. We have shown that mitapivat, a pyruvate kinase activator, improves anemia and ineffective erythropoiesis in Hbbth3/+ mice, a widely used model for β-thal.5 The effects of mitapivat are not limited to the erythroid compartment: mitapivat also modulates DMT1 expression, controlling iron absorption in the duodenum in Hbbth3/+ mice, with an increase of hepcidin related to the improvement in ineffective β-thalassemic erythropoiesis.5 Results from a phase II trial of mitapivat in non–transfusion-dependent β-thal patients previously demonstrated a sustained long-term increase in hemoglobin (Hb ≥1 g/dL) with improvement of hemolysis and ineffective erythropoiesis.6 Here, we asked whether mitapivat might be a potential therapeutic option also for β-thal patients under chronic transfusion regimen. In order to address this question, we exposed female Hbbth3/+ mice (3-4 months of age) to chronic transfusion with or without mitapivat (50 mg/kg twice daily [BID]). Hbbth3/+ mice were treated by oral gavage with mitapivat (50 mg/kg BID) or vehicle for 10 days, and then transfused with 400 μL washed red blood cells at 40-45% hematocrit (Hct)7 (Figure 1A). We defined Hb values ≤10.5 g/dL as the transfusion threshold, corresponding to the reduction of ~50% of post-transfusion Hb values. Normality of data was assessed with the Shapiro-Wilk test. Two-tailed unpaired Student t-test or two-way analysis of variance with Tukey’s multiple comparisons were used for data analyses. Data show values from individual mice and are presented with mean ± standard error of the mean (differences with P<0.05 were considered significant). As shown in Figure 1B, mitapivat-treated β-thal mice exposed to chronic transfusion displayed a greater sustained rise in Hb from baseline compared to vehicle-treated transfused β-thal mice. This resulted in a longer interval between transfusions (13.8±1.0 days in mitapivat- treated β-thal mice vs. 10.5±1.0 days in vehicletreated β-thal mice; Figure 1C). Chronic transfusion resulted in a significant reduction of splenomegaly in both mitapivat- and vehicle-treated β-thal mice (Online Supplementary Figure 1SA) compared to untreated β-thal mice, but spleen iron accumulation was significantly lower in mitapivat-treated β-thal mice when compared to vehicle-treated β-thal mice (Figure 1D). A significant reduction of both bone marrow and spleen ineffective erythropoiesis was observed in all transfused β-thal mice (Figure 1E; Online Supplementary Figure S1B). Of note, mitapivat- treated transfused β-thal mice showed a slight increase of bone marrow erythropoiesis with a trend towards an improvement of maturation index compared to vehicle-treated transfused β-thal mice evaluated at the end of the study.5 This is most likely related to a protective effect of mitapivat on residual bone marrow and spleen erythropoiesis (Figure 1F). Indeed, plasma erythropoietin was lower in mitapivat-treated transfused β-thal mice than in vehicle-treated transfused β-thal mice (Online Supplementary Figure S1C). Since splenic macrophages contribute to both erythrophagocytosis and iron recycling, we evaluated the functional profile of spleen macrophages in the different mouse groups.8 As shown in Figure 1G, flow cytometric analysis of the surface expression of the M1 marker CD80 and the M2 marker CD206 on spleen macrophages (MΦ) revealed that mitapivat promoted a proresolving profile of splenic macrophages in transfused β-thal mice when compared to vehicle-treated transfused β-thal mice (Online Supplementary Figure S2A). This effect was still observed in non-transfused mitapivat-treated mice compared to vehicle-treated β-thal mice (Figure 1G; Online Supplementary Figure S2A). Collectively, these data support the role of mitapivat in reprograming macrophages from proinflammatory to proresolving and repairing the phenotype in β-thal mice with or without chronic transfusion.9 We then evaluated the impact of mitapivat on iron metabolism in transfused β-thal mice. Mitapivat-treated transfused β-thal mice showed lower liver iron accumulation when compared to vehicle-treated transfused β- thal mice (Figure 2A). This might be due in part to the reduction of the transfusion burden but also to the multimodal action of mitapivat, which we previously showed to modulate hepcidin indirectly by the reduction of ineffective erythropoiesis and downregulation of DMT1 expression in the duodenum.5 Indeed, in mitapivat-treated transfused β-thal, we found a significant increase in liver hepcidin/LIC ratio (Figure 2B) and a marked reduction in the percentage of serum transferrin saturation when compared to vehicle-treated transfused β-thal mice (Figure 2C). The reduced transfusion burden observed in mitapivat treated β-thal mice might favorably contribute to the general reduction of iron-overload in β-thal mice exposed to chronic transfusion. Our preclinical results in combination with clinical data from non-transfusion-de- Haematologica | 108 September 2023 2535 LETTER TO THE EDITOR A C E F G D B Continued on following page. Haematologica | 108 September 2023 2536 LETTER TO THE EDITOR pendent β-thal patients treated with mitapivat6 suggest that the increase in the length of time between transfusions with mitapivat treatment may be associated with improvement in the quality of life in patients as well as a decrease in iron-overload-related organ damage. Recent reports in transfusion-dependent β-thal patients have highlighted a correlation between ferritin levels and kidney iron accumulation assessed by magnetic resonance T2* imaging, or in sample analysis from kidney biopsies or autopsy series.10 Kidney iron overload mainly involved the tubular compartment which has been related to chronic anemia and might be reversed by iron chelation.10 In vehicle- treated transfused β-thal mice, we found tubular accumulation of iron, which was significantly reduced in mitapivat-treated transfused animals (Figure 3A). No major difference in creatinine was observed in both β-thal mouse groups exposed to chronic transfusion (Online Supplementary Figure S2B). Previous studies suggest that kidney iron accumulation promotes local oxidative stress, contributing to profibrotic signaling in addition to hypoxia.10,11 MicroRNA (miRNA) let-7b, -c, and -d have been shown to be linked to renal fibrosis throughout the transforming growth factor- β cascade (TGF-β).12 In this study, miRNA let-7b and -d were upregulated in vehicle-treated β-thal mice with or without chronic transfusion (Figure 3B; Online Supplementary Figure S2C), while mitapivat downregulated miRNA let-7b and -d in β-thal mice with or without chronic transfusion (Figure 3B; Online Supplementary Figure S2C). miRNA let-7 have been reported to reduce ATP production by deactivating pyruvate dehydrogenase kinase (PDK).13 Here, we found normalization of the amount of the active form of the TGF-β receptor in β-thal mice treated with mitapivat when compared to vehicle-treated β-thal mice with or without transfusion (Figure 3C). Previously, in β- thal mice, the activation of TGF-β receptor was reduced by chronic transfusion, hypoxia being a trigger of activation of TGF-β receptor.14 Taken together, our data indicate that mitapivat might play a pivotal role in kidney protection by reducing the transfusion burden and iron overload as well as by preserving energy cell metabolism. This might represent an added value of mitapivat as a therapeutic option for patients with β-thal taking iron chelators who develop renal toxicity or chronic kidney disease. Finally, we explored the effects of the co-administration of mitapivat and deferiprone (DFP) on β-thal mice, since iron chelation is part of the gold standard treatment of β- thal patients.1 DFP was administered to Hbbth3/+ mice treated with mitapivat in drinking water at the dosage of either 1.25 or 0.75 mg/mL15 (Online Supplementary Figure S3A). Previously, Casu et al. reported that DFP alone has no effect on hematologic parameters and red cell features in murine β-thal.15 The beneficial effects of mitapivat on murine β-thal anemia was maintained when mitapivat was co-administered with DFP at both dosages, as supported by the stable and sustained increase in Hb and the reduction in circulating erythroblasts compared to baseline values (Online Supplementary Figure S3B, C). In agreement with Matte et al.,5 we found a significant reduction in α-globin membrane precipitates in red blood cells from mitapivat DFP-treated Hbbth3/+ mice compared with vehicle-treated animals (Online Supplementary Figure S3D). Of note, DFP iron chelation efficacy represented by a change in LIC was preserved in β-thal mice treated with both DFP and mitapivat (Online Supplementary Figure S3E). In conclusion, our study shows for the first time that mitapivat improves the transfusion burden and reduces organ iron overload in β-thal mice exposed to a chronic transfusion regimen. We also observed that mitapivat might protect the kidney against profibrotic stimuli related to local iron accumulation by two different mechanisms: the reduction in transfusion requirement and the local modulation of miRNA involved in profibrotic signal- Figure 1. Mitapivat reduces transfusion burden in β-thalassemia mice exposed to chronic transfusion with associated reprogramming of splenic macrophage phenotype. (A) Experimental study design to assess the effects of mitapivat on hematologic phenotype of β-thalassemia (β-thal) mice exposed to chronic transfusion. (B) Hemoglobin (Hb) changes over time in transfused (Tr.) β-thal (Hbbth3/+) mice treated with either vehicle or mitapivat (50 mg/kg twice daily [BID]) shown as single animals (n=3 vehicle- treated mice; n=4 mitapivat-treated mice). Grey dotted line shows the transfusion threshold (10.5 g/dL). (C) Transfusion time intervals in β-thal (Hbbth3/+) mice treated with either vehicle or mitapivat (50 mg/kg BID). Data are presented as means ± standard error of the mean (SEM) (n=3 vehicle-treated mice; n=4 mitapivat-treated mice); #P<0.05 compared to vehicle-treated transfused β-thal mice. (D) Iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in spleen from Hbbth3/+ mice treated with either vehicle or transfusion plus vehicle or transfusion plus mitapivat. One representative image from 3 with similar results. Left panel: quantification of iron staining in spleen. Data are mean ± SEM (n=3). *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. (E) Flow cytometric analysis (CD44+Ter119+ and cell size markers, see also the Online Supplementary Figure S2) of bone marrow and spleen from Hbbth3/+ mice exposed to either vehicle or to chronic transfusion with and without mitapivat treatment (see also Matte et al.5). Data are mean ± SEM (n=3-4). *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. (F) Maturation index as ratio between pop II (Baso E.) and pop IV (Ortho E.) in bone marrow and spleen from Hbbth3/+ mice treated with either vehicle or exposed to chronic transfusion with or without mitapivat, analyzed by flow cytometry. Data are mean ± SEM (n=3-4). (G) Flow cytometric quantification of M1 (CD80) and M2 (CD206) expression on spleen macrophage cell surface from wild-type (WT) or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment. Spleen macrophages (MΦ) were isolated with the GentleMACS cell dissociator (Miltenyi Biotech, Germany). MΦ were identified and gated as CD45+/F4/80+ cells. Anti-CD45 PE-Cy5.5, F4/80 PE, CD206 PerCP-Cy5.5 and CD80 were from BioLegend, USA. Data are mean ± SEM (n=3-4). MFI: mean fluorescence intensity; RBC: red blood cells. Haematologica | 108 September 2023 2537 LETTER TO THE EDITOR A B C Figure 2. Mitapivat-treated transfused β-thalassemia mice show reduced liver iron accumulation and improved iron homeostasis. (A) Left and central panels: iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in liver from wild-type (WT) and Hbbth3/+ mice treated with either vehicle or transfusion (Tr.) or transfusion plus mitapivat. One representative image from 5 with similar results. Right panel: quantification of iron staining in liver. Data are mean ± standard error of the mean (SEM) (n=5). °P<0.05 compared to WT, *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused (Tr.) Hbbth3/+ mice. (B) Liver mRNA expression normalized over liver iron concentration (LIC) as determined using the bathophenanthroline method. Data are presented as means ± SEM (n=3). #P<0.05 compared with vehicletreated transfused Hbbth3/+ mice. (C) Transferrin saturation in Hbbth3/+ mice treated with either vehicle or transfusion or transfusion plus mitapivat. Transferrin saturation was calculated as the ratio between serum iron and total iron binding capacity, using the Total Iron Binding Capacity Kit (Randox Laboratories, UK) and 50 mL of serum, according to the manufacturer’s instructions. Data are presented as means ± SEM (n=3). *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. RBC: red blood cells. Haematologica | 108 September 2023 2538 LETTER TO THE EDITOR A B C Figure 3. In transfused β-thalassemia mice, mitapivat reduces kidney iron accumulation and downregulates profibrotic kidney miRNA let-7 expression. (A) Upper panels: iron staining (Perl’s Prussian blue is a semi-quantitative method to assess organ iron accumulation) in kidney from wild-type (WT) and Hbbth3/+ mice treated with either vehicle or transfusion (Tr.) or transfusion plus mitapivat. One representative image from 3-6 with similar results. Lower panels: quantification of iron staining in kidney. Data Continued on following page. Haematologica | 108 September 2023 2539 LETTER TO THE EDITOR ing. Finally, the observed reprograming of spleen macrophages toward a proresolving phenotype might represent an added value to the known improvement of ineffective erythropoiesis induced by mitapivat in β-thal mice.5 Thus, the beneficial effects of mitapivat in β-thal mice exposed to chronic transfusion support its use as a potential new therapeutic tool in clinical management of thalassemic patients under chronic transfusion regimen. Authors Alessandro Mattè,1 Penelope A. Kosinski,2 Enrica Federti,1 Lenny Dang,2 Antonio Recchiuti,3 Roberta Russo,4 Angela Siciliano,1 Veronica Riccardi,1 Anne Janin,5 Matteo Mucci,3 Christophe Leboeuf,5 Achille Iolascon,6 Carlo Brugnara7 and Lucia De Franceschi1 1University of Verona and AOUI Verona, Verona, Italy; 2Agios Pharmaceuticals, Inc., Cambridge, MA, USA; 3Deptartment of Medical, Oral and Biotechnology Science, “G. d’Annunzio” University of Chieti, Chieti, Italy; 4Dipartmento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, Naples, Italy; 5University Diderot of Paris, Paris, France; 6CEINGE - Biotecnologie Avanzate Franco Salvatore, Naples, Italy and 7Department of Laboratory Medicine, Harvard Medical School, Boston Children's Hospital, Boston, MA, USA Correspondence: L. DE FRANCESCHI - [email protected] https://doi.org/10.3324/haematol.2022.282614 Received: December 20, 2022. Accepted: February 7, 2023. Early view: February 16, 2023. ©2023 Ferrata Storti Foundation Published under a CC BY-NC license Disclosures LDF received research funding from Agios during 2015-2022. LD and PAK are Agios employees and stockholders. All other authors have no conflicts of interest to disclose. Contributions LDF, CB, AM and AI designed and carried out research and wrote the paper. PAK, LD and CB critically revised data and wrote the paper. AM, EF, AS and VR carried out cytokine FACS analysis, immunoprecipitation assays and ELISA analysis. RB carried out molecular analysis. EF revised the paper. MM and AR carried out miRNA analysis, analyzed the data and wrote the paper. CL and AJ performed pathology analysis and analyzed data. Funding This study was supported by an Agios Pharmaceuticals, Inc. research collaborative grant to LDF. Editorial assistance was provided by Avant Healthcare, LLC and Excel Medical Affairs, Horsham, UK, supported by Agios. Data-sharing statement All the data and protocols are stored in the Nas Synology DS216se Hard Disk, located at the University of Verona, Verona, Italy and will be made available on request. Please direct requests for original data to the corresponding author. are mean ± standard error of the mean (SEM) (n=3-6). *P<0.05 compared with vehicle Hbbth3/+ mice and #P<0.05 compared with vehicle-treated transfused (Tr.) Hbbth3/+ mice. (B) Relative expression of miRNA let-7b and -7d in kidneys from WT or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment. Small RNA was isolated from frozen kidneys using a silica spin column-based Quick-RNA kit (Zymo Research), quantified with a UV NanoPhotometer (Implen), and reverse transcribed with the qScript microRNA cDNA Synthesis for RT-PCR (QuantaBio). For real time polymerase chain reaction (RT-PCR) analysis of let-7b and let-7d miRNA, 3 ng of cDNA were used as a template in reaction mixtures (10 mL final volume) including a PowerUp SYBR Green Master Mix (5 mL, Applied Biosystems), miRNA-specific forward and universal reverse primers (1 mL each, miRCURY assays, Qiagen), and PCR-grade water. The expression of the indicated mRNA was quantitated by the comparative ΔCt method. RNU6-1 was used as control for normalization. Data are mean ± SEM (n=3-4). *P<0.05. **P<0.01. ***P<0.001. (C ) Phospho-tyrosine immunoprecipitation of kidneys from WT or Hbbth3/+ mice exposed to either vehicle or mitapivat or to chronic transfusion with and without mitapivat treatment, using anti-phospho-tyrosine specific antibodies (IP: PY, clone PY99 from SCBT, Santa Cruz, CA and clone 4G10 from Merck KGaA, Darmstadt, Germany), revealed with anti-TGF-β receptor (Rec) specific antibody. GAPDH in whole-cell lysate (WCL) is used as loading control. One representative gel from 4 others with similar results is presented. Blots were developed using the Luminata Forte Chemiluminescent HRP Substrate from Merck Millipore (Armstadt, Germany), and images were acquired with the Alliance Q9 Advanced imaging system (Uvitec, UK). Densitometric analysis of immunoblots is shown on the right. Data are mean ± SEM (n=4). °P<0.05 compared to WT; *P<0.05 compared with vehicle Hbbth3/+ mice, #P<0.05 compared with vehicle-treated transfused Hbbth3/+ mice. RBC: red blood cells. References 1. Taher AT, Musallam KM, Cappellini MD. Beta-Thalassemias. N Engl J Med. 2021;384(8):727-743. 2. De Franceschi L, Bertoldi M, Matte A, et al. Oxidative stress and β-thalassemic erythroid cells behind the molecular defect. Oxid Med Cell Longev. 2013;2013:985210. 3. Rivella S. β-thalassemias: paradigmatic diseases for scientific Haematologica | 108 September 2023 2540 LETTER TO THE EDITOR discoveries and development of innovative therapies. Haematologica. 2015;100(4):418-430. 4. Locatelli F, Thompson AA, Kwiatkowski JL, et al. Betibeglogene autotemcel gene therapy for non-beta(0)/beta(0) genotype beta-Thalassemia. N Engl J Med. 2022;386(5):415-427. 5.Matte A, Federti E, Kung C, et al. The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a betathalassemia mouse model. J Clin Invest. 2021;131(10):e144206. 6. Kuo KHM, Layton DM, Lal A, et al. Safety and efficacy of mitapivat, an oral pyruvate kinase activator, in adults with nontransfusion dependent alpha-thalassaemia or beta-thalassaemia: an open-label, multicentre, phase 2 study. Lancet. 2022;400(10351):493-501. 7. Park SY, Matte A, Jung Y, et al. Pathologic angiogenesis in the bone marrow of humanized sickle cell mice is reversed by blood transfusion. Blood. 2020;135(23):2071-2084. 8. Ramos P, Casu C, Gardenghi S, et al. Macrophages support pathological erythropoiesis in polycythemia vera and betathalassemia. Nat Med. 2013;19(4):437-445. 9. Galvan-Pena S, O'Neill LA. Metabolic reprograming in macrophage polarization. Front Immunol. 2014;5:420. 10. Demosthenous C, Vlachaki E, Apostolou C, et al. Betathalassemia: renal complications and mechanisms: a narrative review. Hematology. 2019;24(1):426-438. 11. Musallam KM, Taher AT. Mechanisms of renal disease in betathalassemia. J Am Soc Nephrol. 2012;23(8):1299-1302. 12. Hong S, Lu Y. Omega-3 fatty acid-derived resolvins and protectins in inflammation resolution and leukocyte functions: targeting novel lipid mediator pathways in mitigation of acute kidney injury. Front Immunol. 2013;4:13. 13.Ma X, Li C, Sun L, et al. Lin28/let-7 axis regulates aerobic glycolysis and cancer progression via PDK1. Nat Commun. 2014;5:5212. 14. Chou YH, Pan SY, Shao YH, et al. Methylation in pericytes after acute injury promotes chronic kidney disease. J Clin Invest. 2020;130(9):4845-4857. 15. Casu C, Aghajan M, Oikonomidou PR, et al. Combination of Tmprss6- ASO and the iron chelator deferiprone improves erythropoiesis and reduces iron overload in a mouse model of beta-thalassemia intermedia