Therapeutic potential of haptoglobin in a murine model of sickle cell anemia

Bianca Cristina dos Santos; Magnun Nueldo Nunes dos Santos; Gisele Audrei Pedroso; Beatriz Benedetti de Oliveira; Dulcinéia Martins de Albuquerque; Nathalia Rocha de Oliveira; Daniela Cagnoto Noronha; Fernando Ferreira Costa; Marcus Alexandre Finzi Corat

doi:10.1371/journal.pone.0357180

. 2026 Sep 18;21(9):e0357180. doi: 10.1371/journal.pone.0357180

Therapeutic potential of haptoglobin in a murine model of sickle cell anemia

Bianca Cristina dos Santos ^1,^#, Magnun Nueldo Nunes dos Santos ^2,^#, Gisele Audrei Pedroso ³, Beatriz Benedetti de Oliveira ³, Dulcinéia Martins de Albuquerque ⁴, Nathalia Rocha de Oliveira ¹, Daniela Cagnoto Noronha ¹, Fernando Ferreira Costa ⁴, Marcus Alexandre Finzi Corat ^1,^*

Editor: Fábio Henrique Silva⁵

PMCID: PMC13588521 PMID: 42758770

Abstract

Chronic hemolytic anemia found in patients with sickle cell disease promotes an excess of free extracellular and intravascular hemoglobin, triggering a series of deleterious effects such as endothelial dysfunction, oxidative stress, and alterations in vascular tone, which lead to clinical complications. In this work, we evaluated the induced expression of haptoglobin in hepatocytes in vivo, the organism’s first line of defense to control the presence of free Hb in circulation, which becomes saturated in conditions of chronic hemolysis, such as sickle cell disease. Through gene therapy applied in a murine model of sickle cell anemia, we aimed to investigate whether this approach could interfere with disease progression and promote an improvement in the animals’ pathophysiological condition. The application of this treatment in the sickle cell anemia animal model resulted in significant therapeutic effects, including improvement of hematological parameters, reduction of the compensatory erythropoietic response, and attenuation of clinical signs such as splenomegaly. These results indicate that haptoglobin may act indirectly in the prevention of hemolysis, suggesting a new therapeutic approach.

Introduction

Sickle cell disease (SCD) is a genetic hemoglobinopathy that causes a morphological and functional variation in red blood cells. Patients present a point mutation responsible for the substitution of a base in codon 6 of the β-globin gene. In this case, an adenine is replaced by a thymine in the sequence, resulting in the substitution of the amino acid glutamate by valine, leading to the formation of hemoglobin S (HbS). Under hypoxic conditions, HbS polymerizes, generating a cellular deformation that makes the red blood cells elongated and sickle-shaped. This hinders their passage through the capillaries, leading to vascular obstructions, tissue ischemia, and frequent pain crises [1–4].

These sickled cells also have a reduced lifespan compared to normal erythrocytes, and this premature destruction causes a chronic hemolytic anemia condition. The accelerated destruction of erythrocytes is described as hemolysis and is a central component of the disease’s pathophysiology. During hemolysis, there is a massive release of free hemoglobin into the bloodstream. This extracellular hemoglobin, when not properly captured by natural clearance mechanisms, can trigger a series of deleterious effects, such as endothelial dysfunction, oxidative stress, and changes in vascular tone, further worsening the clinical complications of SCD [4].

The first line of defense to control the presence of free Hb in the circulation is through the action of haptoglobin (Hp). Hp is a plasma protein synthesized by the liver and was located on the long arm of chromosome 16 in 1969 [5]. It has two main alleles (Hp1 and Hp2), which by dimerization determine three distinct phenotypes (Hp1−1; Hp2−1; and Hp2−2) [6].

Besides its origin and genetic variations, the main function of haptoglobin is to bind specifically and with high affinity to hemoglobin released during intravascular hemolysis, forming a haptoglobin-hemoglobin complex (Hp -Hb) [7,8] By binding to free Hb, this interaction prevents it from circulating to organs like the kidneys, where its activity may lead to cellular injury. This is because the formed complex is rapidly recognized by macrophages in the liver and spleen, being removed from circulation through the rapid binding of the Hb-Hp complex to the CD163 receptor found on macrophages, which internalize the free hemoglobin and initiate proteolytic degradation of hemoglobin and heme catabolism, preventing the oxidation of Fe2+ [7,9]. This mechanism is essential to prevent the toxicity associated with free hemoglobin.

In addition to the hemolytic protection systems related to haptoglobin, there are also more downstream protective mechanisms in the iron oxidation process, such as the hemopexin/ heme oxygenase/biliverdin reductase pathway, which primarily acts on heme degradation, and transferrin, which acts directly on iron already oxidized to Fe3 + , preventing its further oxidation to the more toxic Fe4 + state [10,11]. However, under pathological conditions with sustained intense and chronic hemolysis, leading to continuously increasing levels of free hemoglobin, as occurs in SCD, these clearance systems become saturated, resulting in the progressive accumulation of free hemoglobin in the circulation. This leads to the oxidation of Fe2+ to Fe4 + , worsening the vascular and inflammatory complications characteristic of the disease [12,13].

With the aim of enhancing the first line of hemolytic defense, this study chose to promote the overexpression of haptoglobin 1 in order to evaluate the organism’s response to this treatment during the development of sickle cell disease. The choice of haptoglobin phenotype Hp1 was based on literature evidence demonstrating the greater efficiency of this phenotype in neutralizing the deleterious effects of free hemoglobin. Studies indicate that Hp1−1 exhibits higher antioxidant capacity compared to Hp2−2, promoting the formation of more stable Hb-Hp complexes with more efficient clearance via endocytosis. In contrast, complexes formed with Hp2−2 are associated with increased generation of reactive oxygen species, thereby enhancing the potential for oxidative damage to lipids, proteins, and DNA. Based on this evidence, the hypothesis is proposed that increasing circulating levels of haptoglobin may favor the formation of the Hb-Hp complex and, consequently, promote a more efficient removal of free hemoglobin. This approach could minimize the harmful effects of chronic hemolysis and thus represent a promising alternative to reduce the severity of the clinical manifestations of the disease. Therefore, considering the context of chronic hemolysis observed in sickle cell anemia, Hp1 was considered more appropriate as a therapeutic strategy due to its greater efficiency in reducing oxidative stress and its potential tissue-protective effects.

Materials and methods

Bone marrow transplantation to generate SCD mouse models

In this study, a mouse model with SCD (SCM) was adopted, generated by transplanting bone marrow from Berkeley SCM model, which is homozygous sickle cell mice STOCK (Hbatm1Paz Hbbtm1Tow Tg(HBA-HBBs)41Paz/J), as described by Reddy [14,15]. This approach allowed for the generation of mice with hematological characteristics compatible with SCD, through the replacement of the recipients’ hematopoietic stem cells with cells from donors carrying the sickle cell genotype. The bone marrow donor mice used in this study belong to the Berkeley model, a widely used humanized model of sickle cell disease that predominantly expresses human hemoglobin S. Additionally, the irradiated recipient animals belong to the species Mus musculus, C57BL/6J strain. All procedures involving animal models were approved by the Animal Use Ethics Committee (CEUA) of the University of Campinas (UNICAMP) – São Paulo, Brazil (6391–1/2024). Six animals were transplanted in each experimental group. In both groups, deaths occurred, with one death recorded in the treated group and two in the control group. In the treated group, one animal showed a high level of chimerism (~95%), while another presented a very low level and was therefore excluded from the analysis. Thus, three animals with intermediate levels of chimerism (mean approximately 75%) were included in the analysis. In the control group, animals with chimerism levels comparable to those observed in the treated group were selected, totaling three animals for analysis..

To obtain the donor cells, a sickle cell mouse was anesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg), ensuring adequate sedation, analgesia, and muscle relaxation for the procedure. For euthanasia, a high anesthetic dose (300 mg/kg ketamine and 30 mg/kg xylazine) was administered, followed by cervical dislocation, in accordance with the guidelines of the Animal Ethics Committee (CEUA) and CONCEA. In addition, measures were implemented throughout the entire experiment to minimize animal suffering, including continuous monitoring, appropriate anesthesia and analgesia protocols, and procedures conducted by a trained team, in compliance with the 3Rs principles. After euthanasia, the long bones (femur and tibia) from the hind limbs were collected. Following disinfection with 70% ethanol, the bones were washed with DMEM medium, which was also used for bone marrow extraction using insulin syringes. The cell suspension was homogenized and subjected to red blood cell lysis using ammonium-chloride-potassium (ACK) solution in order to isolate hematopoietic progenitor cells.

The recipient mice were previously subjected to irradiation with a dose of 900 rads of gamma radiation to eliminate bone marrow cells and allow for the integration of the transplanted cells. The donor cell infusion was performed intravenously at a dose of 3 × 10⁶ cells in 0,1 mL, using the orbital plexus as the administration route—an effective and less invasive method widely used in experimental models.

The choice of the transplantation methodology was due to the limited availability and fragility of established transgenic SCD models, which restrict their application in large-scale experimental studies. Moreover, the transplanted model allows for the evaluation of specific pathophysiological aspects and the testing of therapeutic interventions in a controlled manner, contributing to the advancement of research on the disease

Haptoglobin cloning using the pGEM-T Easy vector

The cloning of the haptoglobin protein was performed using recombinant DNA technology. The cDNA corresponding to the messenger RNA (mRNA) of human haptoglobin was obtained by reverse transcription followed by PCR (RT-PCR), using specific primers designed on the Benchling platform. The reaction was carried out in a final volume of 25 µL, containing 12.5 µL of CloneAmp mix (including enzyme, buffer, and dNTPs), 1 µL of each primer, 5 µL of cDNA, and 5.5 µL of water.

The amplified fragment was inserted into the pGEM-T Easy vector (Promega), which has 3’ thymine overhangs, facilitating ligation with PCR-generated products. The ligation reaction was carried out in a 10 µL volume containing 5 µL of T4 DNA ligase buffer, 1 µL of the vector, 3 µL of the PCR product, and 1 µL of T4 DNA ligase enzyme, incubated overnight at 4 °C.

After ligation, the recombinant vector was transformed into competent Escherichia coli cells, which were cultured in selective medium containing ampicillin. The resulting colonies were screened by PCR to identify clones containing the insert.

Subsequently, the selected coding fragments were subcloned into the Lego tdTomato vector, aiming at gene expression of haptoglobin with the tdTomato reporter gene assisted by the P2A cleavage peptide.

Cloning of haptoglobin into the Lego-tdTomato plasmid

After the initial cloning of the haptoglobin coding sequence into the pGEM-T Easy vector, the region of interest was subcloned into the lego tdTomato expression plasmid, derived from pEGFP-C1. Unlike the original vector, this plasmid expresses the red fluorescent protein tdTomato, allowing the tracking of transfected cells through fluorescence microscopy. The expression vector was designed for bicistronic expression of haptoglobin, concomitant with the tdTomato reporter protein separated by the P2A cleavage peptide. Expression control was carried out by the SFFV (Spleen Focus-Forming Virus) promoter, known to drive strong gene expression, to promote overexpression of the haptoglobin transgene. The human haptoglobin sequence (GenBank: NG_012651.1) was inserted into the lego tdTomato plasmid using restriction enzymes and T4 DNA ligase.

After confirmation of cloning through analysis of bacterial growth, the recombinant vector containing haptoglobin was subjected to sequencing and was also introduced into HEK 293-T cells by lipid-based transfection to verify gene expression. These cells, derived from human embryonic kidney, were chosen because they are widely used in gene expression experiments due to their high transcription and translation rates, as well as their ease of culture and handling.

The cells were cultured until they reached appropriate confluence for transfection. The expression of the tdTomato protein was evaluated by fluorescence microscopy, with red fluorescence emission indicating successful expression of the construct containing both proteins. This step allowed the selection of transfected cells and the validation of the gene expression system for subsequent phases of the experiment.

Sequencing

Genetic sequencing was used to validate the results obtained by PCR and to confirm the integrity of the sequences of interest. The technique employed was Sanger sequencing, which is based on the extension of DNA fragments from specific primers, with the incorporation of deoxynucleotide triphosphates (dNTPs), generating products of different sizes that allow the reading of the nucleotide sequence.

Recombinant plasmids were extracted using the QIAprep Spin Miniprep kit (QIAGEN) in order to eliminate impurities such as residual dNTPs, free primers, enzymes, and salts. Quantification and purity assessment of the samples were performed by spectrophotometry using a nanodrop, considering absorbance at 260 nm as an indicator of nucleic acid concentration.

For sequencing, specific oligonucleotides targeting the regions of interest were designed using the Benchling platform. Data interpretation was conducted based on the analysis of chromatograms generated by the sequencing service, using Chromas software.

Introduction of the plasmid into animals

Assays were conducted for the transfection of the Hapto-tdTomato plasmid. The efficiency of gene expression was determined based on the fluorescence observed in hepatic cells after transfection. The plasmid was prepared for animal transfection via lipid-based transfection. Ten micrograms (10 µg) of plasmid were mixed with the commercial reagent JetOPTIMUS solution according to the manufacturer’s instructions, and administered to mouse models transplanted one week earlier with sickle hematopoietic progenitor cells, via the retro-orbital plexus with 100 µL of the solution. The animals were euthanized after 60 days for analysis of gene expression by microscopy to verify the expression of the tdTomato fluorescent protein in the hepatic parenchyma, as well as for tissue collection for pathological analysis.

Pathological analysis

To evaluate the effects of the proposed treatment in a sickle cell anemia experimental model, comparative pathological analyses were conducted, in a double- blind manner, between control and treated groups. The methodological approaches included quantification of hemoglobins by HPLC, complete blood count, assessment of spleen weight, and histological analysis of target organs. These evaluations enabled the investigation of hematological, morphological, and tissue alterations associated with the disease and the treatment. Data were analyzed using GraphPad Prism software, applying the unpaired t-test with a significance level of 5%. Results were presented in graphs and expressed as mean ± standard deviation.

1. High-performance liquid chromatography

The qualitative and quantitative determination of hemoglobin fractions in peripheral blood was performed by cation-exchange high-performance liquid chromatography (HPLC), using the automated Variant II system (Bio-Rad). Engraftment was confirmed using this approach. Peripheral blood samples were collected via submandibular puncture and automatically aspirated, injected into the analytical system, and applied to a cation-exchange column (stationary phase). A programmed buffer gradient (mobile phase), driven by dual-piston pumps, increased the ionic strength and promoted the separation of different hemoglobin fractions based on their ionic interactions with the column. The eluted fractions were detected by photometry at 415 nm, identified by their characteristic retention times, and quantified by the area under the elution peaks.

2. Complete Blood Count (CBC)

The complete blood count was performed on anticoagulated whole blood samples using the automated hematology analyzer XN-9000 (Sysmex). This system employs four detection methods: the RF/DC (radiofrequency/direct current) method, which evaluates cell volume through direct current resistance and intracellular density based on variations in radiofrequency resistance; hydrodynamic focusing, which ensures cell alignment in the laminar flow, minimizing artifacts and false impulses during counting; flow cytometry with a semiconductor laser, used to analyze blood cells and particles through light emission by stained RNA, DNA, and proteins, allowing the separation of cell populations using scatter plot analysis; and the SLS-hemoglobin method, which uses sodium lauryl sulfate (SLS), free of cyanide, to lyse erythrocytes and form SLS-HGB complexes, whose concentration is determined by photometry using a monochromatic LED and detection by a photosensor.

3. Spleen weight

For the analysis of spleen weight, the animals were euthanized according to a protocol approved by the ethics committee, and the spleen was carefully dissected, removed, and immediately weighed on a precision analytical balance. The data obtained were used for comparison between the experimental groups, considering both the absolute weight of the organ and its ratio to the total body weight of the animals. This analysis aimed to evaluate splenic alterations associated with the sickle cell condition and the effects of the proposed treatment.

4. Histopathological assessment

For histological analysis, the animals were euthanized according to a protocol approved by the ethics committee. The organs of interest were collected, fixed in 10% buffered formalin solution, routinely processed, embedded in paraffin, and sectioned using a microtome at a thickness of 4 μm. The slides were stained with hematoxylin and eosin (HE) and analyzed under a light microscope. Histological evaluation was performed comparatively between groups, considering morphological parameters and the presence of tissue alterations associated with sickle cell anemia and the effects of the experimental treatment.

Results

To investigate the contribution of haptoglobin overexpression in an organism with chronic hemolysis during the development of sickle cell anemia, we initially constructed a strong expression vector to express haptoglobin and a reporter protein to facilitate its tracking (Fig 1). The vector is driven by the strong promoter of the Spleen Focus-Forming Virus – SFFV, producing a single messenger RNA with bicistronic expression, first of the haptoglobin protein and second of the TdTomato reporter gene (red fluorescent) in frame. This ensures efficient expression and visualization of gene expression in cells. The SFFV promoter is known for its strong constitutive activity, ensuring robust and stable expression. Next, the coding sequence of human haptoglobin was inserted from a hepatic cDNA library. To allow simultaneous but independent expression of a fluorescent marker, the P2A cleavage peptide was used, which promotes ribosomal skipping during translation, allowing the production of two separate proteins from a single transcript. The chosen marker was tdTomato, a fluorescent gene derived from DsRed, which emits intense and stable red fluorescence. The presence of tdTomato in the construct allows for the visual identification and tracking of cells expressing the haptoglobin gene, facilitating the selection and functional analysis of transfectants. 293-T cells were used to validate the construct expression system.

Fig 1 — The plasmid contains the SFFV promoter, the human haptoglobin coding sequence, the P2A cleavage site, and the td-Tomato fluorescent marker, allowing bicistronic expression and visualization of transgene expression in 293-T cells.

To ensure greater fidelity of the haptoglobin sequence, the sequence cloned into the vector was sequenced (Fig 2). The sequence showed 99,74% identity with the reference gene sequence NG_012651.1; 99.79% with the mRNA sequence NM_005143 using the BLASTN tool, and 99.69% with the reference protein sequence NP_005134 using BLASTP. Comparison with the reference sequence revealed high identity, with only six-point variations, three of which resulted in amino acid substitutions. The observed changes included: substitution of aspartic acid with asparagine, which may impact electrostatic interactions and the protein’s catalytic activity; replacement of lysine with glutamic acid, altering the charge profile of the region and potentially influencing interactions with substrates or other biomolecules; and substitution of leucine with proline, a change that may affect the local conformation of the polypeptide chain due to proline’s structural rigidity. Despite these modifications, the obtained sequence maintains high similarity to the reference, and the point mutations do not appear to compromise the expected functionality of the protein.

After the in vitro analyses, the application of the plasmid was evaluated in a mouse model of sickle cell anemia. The methodology adopted had been previously standardized using cationic liposomes for hepatic transfection of the plasmid via intravenous administration through the orbital plexus with 100 µL of buffered saline solution containing 10 µL of liposomes and 10 µg of the construct. After 26 days, microscopy revealed red fluorescence in multiple hepatic regions (Fig 3), confirming the efficacy of the protocol. The 26-day time point for the analyses was selected based on logistical aspects related to experimental organization and equipment availability. The plasmid containing the cloned sequence was introduced into animals developing SCD one week after transplantation, and PBS was used in animals assigned as SHAM. After 60 days of disease progression, the animals were sacrificed, and physiological samples of blood and organs such as liver, spleen, and kidney were collected for analysis of clinical parameters to validate the treatment. Among the analyzed parameters, hemoglobin levels, hematocrit, and reticulocytes stand out, as they are important indicators of the animals’ hematological status (Fig 4).

Fig 3 — Hepatic red fluorescence observed 26 days after retro-orbital injection of the hapto-tdTomato plasmid confirms efficient delivery and expression.

Hematological analysis (Fig 4) revealed deleterious and expected alterations in animals with SCD, which were partially protected by treatment with haptoglobin transfection. The hematocrit of SCD animals ranged from 16.80 to 23.45%, indicating severe anemia. In the treated group, the values increased significantly (26.70 to 30.10%; p = 0.0013), although still below the physiological range for mice (35–50%), undoubtedly suggesting improved stability and/or production of erythrocytes. Hemoglobin levels also increased with treatment (8.50–11.0 g/dL) compared to the SCD group (4.60–9.70 g/dL), with a significant difference (p = 0.0236), lifting the animals out of a severe anemia condition. Due to limited blood volume, free hemoglobin and plasma haptoglobin could not be reliably quantified, as haptoglobin levels were below the detection limit of the assay.. Following this physiological improvement profile, the reticulocyte count, elevated in the SCA group (9.225–10.193 × 10⁵ cells), was reduced in the treated group (5.408–7.205 × 10⁵ cells), with a statistically significant difference (p = 0.0015). This reduction suggests a decrease in compensatory erythropoiesis, reflecting improved erythrocyte survival.

Platelet counts were also significantly elevated in the treated group, indicating a partial recovery of hematopoietic function, especially in myeloid lineage. In contrast, white blood cell (WBC) counts did not differ significantly between groups, with high inter-sample variability observed. Differential leukocyte counts were not performed in this study, limiting a more detailed assessment of specific inflammatory cell populations such as neutrophils. Similarly, no significant changes were observed in %HbS levels between treated and untreated groups, indicating that the therapeutic effects observed are likely related to modulation of hemolysis rather than alterations in hemoglobin composition. Finally, a significant lower size in the relative spleen size (weight/body weight) was observed in the treated animals (p = 0.0009), suggesting decreased sequestration and erythrocyte destruction. The SCD group showed splenomegaly, characteristic of chronic hemolysis, whereas the treatment protected the organism from spleen enlargement, indicating an attenuation of the functional overload (Fig 4).

Complementarily, histological evaluation revealed significant tissue impairment in the target organs of sickle cell anemia, with partial impact from the treatment. In the kidneys of animals with SCD, red blood cell accumulation, edema, and vascular dilation were observed, especially in the cortical region, along with congestion in the medullary region. These alterations reflect microvascular obstruction induced by sickling of red blood cells. In the treated group, although the presence of red blood cells was still evident, the renal structures did not show worsening, indicating a possible partial containment of the damage (Fig 5).

Fig 5 — Kidneys from untreated SCD mice showed red blood cell accumulation, edema, and vascular congestion; treated mice showed partial structural preservation. Spleens from untreated mice presented white pulp disorganization and intense inflammation; treated mice showed improved architecture. Livers from untreated mice displayed sinusoidal dilation, inflammatory infiltration, and deformed red blood cell accumulation; treated mice had better sinusoid organization and signs of reduced hepatic stress.

Hepatic histology of animals with SCD revealed sinusoidal dilation, inflammatory infiltration, and accumulation of deformed red blood cells, compromising intrahepatic circulation. In treated animals, despite the persistence of red blood cells in the vessels, better organization of the sinusoids was observed, along with indications of reduced hepatic stress.

These histopathological findings confirm the systemic impact of sickle cell anemia on multiple organs and indicate that the tested treatment was able to promote partial improvements in inflammatory and structural aspects, especially in the spleen and liver, although it was insufficient to fully reverse the alterations associated with the sickle cell condition.

Discussion

Chronic hemolysis in Sickle Cell Disease leads to the persistent accumulation of extracellular and intravascular free hemoglobin, promoting endothelial dysfunction, oxidative stress, and disturbances in vascular homeostasis that contribute directly to the clinical manifestations of the disease. In the present study, we focused on haptoglobin, the major endogenous scavenger of circulating free hemoglobin, whose physiological buffering capacity becomes overwhelmed under chronic hemolytic conditions such as sickle cell disease. To further get evidence of the protective role of haptoglobin in this context, we induced the in vivo hepatic expression of haptoglobin-1 in a murine model developing sickle cell disease generated through bone marrow transplantation using the Berkeley SCD model. By increasing circulating haptoglobin levels and favoring the formation of Hb–Hp complexes, we investigated whether enhanced hemoglobin clearance could attenuate the deleterious consequences associated with chronic hemolysis.

Transgenic models have been widely used to mimic human diseases as sickle cell disease [16]. The Berkely SCD model used in this work reproduces relevant phenotypic characteristics of SCD, including alterations in red blood cell morphology and impaired tissue oxygenation, and are also useful for evaluating therapeutic interventions [15,17].

We performed irradiated murine models subjected to SCD bone marrow transplantation. The irradiation eliminates the recipient’s hematopoietic cells, promoting the integration of donor-derived SCD model’s stem cells [14]. Following transplantation, hematological, molecular, and functional parameters are evaluated.

The transplanted models were considered to exhibit severe sickle cell anemia phenotype 60 days post-transplantation, with an HbS proportion above 70%, as described by Iannone et al [18]. Wild-type red blood cells, gamma-irradiated erythrocytes exhibit a significantly reduced lifespan, estimated at approximately 10 days. Therefore, at 60 days post-transplantation, animals predominantly present erythrocytes derived from the transplanted bone marrow, expressing physiological and molecular features consistent with sickle cell anemia, regarding animals with more than 60% of HbS post transplanted [18]. Additionally, in the absence of transplantation, animals do not survive beyond 7–10 days due to the lack of functional erythrocytes.

Regarding the treatment of SCD, several approaches have been proposed to improve patients’ clinical outcomes. Hydroxyurea remains the standard therapy, demonstrating efficacy in reducing complications across different age groups, although its clinical acceptance remains limited, which drives the search for alternative strategies [19,20]. In this context, novel therapeutic targets include inflammation, cell adhesion, hemolysis, and endothelial dysfunction, and new drugs have been approved, while numerous clinical trials are investigating agents with complementary mechanisms [19]. Therapies such as L-glutamine, voxelotor, and crizanlizumab have shown benefits by reducing oxidative stress, improving hemoglobin oxygen affinity, and decreasing vaso-occlusive events [19]. In parallel, more advanced strategies include hematopoietic stem cell transplantation and gene therapy, with gene addition and gene editing approaches capable of correcting mutations or reactivating fetal hemoglobin production, the latter being considered a promising long-term curative alternative [21].

In this context, haptoglobin (Hp) has been widely investigated due to its role in binding and clearing free hemoglobin, thereby contributing to the reduction of hemolysis-associated toxicity. Studies indicate that increased Hp levels can neutralize circulating toxic components and prevent complications such as albuminuria [22]. In addition, Hp has been proposed as a biomarker in conditions such as transplant-associated thrombotic microangiopathy, having been identified as a promising protein in proteomic analyses [23]. Experimental evidence demonstrates that Hp can reduce vascular injury, vaso-occlusive crises, and hemoglobin accumulation in tissues [24].The Hp–Hb complex acts as a scavenger of free hemoglobin and heme, reducing oxidative stress and inflammation [25]. This complex is taken up by macrophages via the CD163 receptor, activating heme oxygenase-1 and promoting cytoprotective and anti-inflammatory effects [7]. Furthermore, Hp plays a relevant role in preventing vascular damage associated with chronic hemolysis [26],as well as in immune modulation, renal protection, and its association with various clinical conditions [27]. Despite all these characteristics, the high level of haptoglobin sequestration in a chronic hemolytic disease may affect conclusive responses in the organism. Therefore, to get insights regarding Hp effects into the chronic hemolytic background, we have analyzed a mouse model on SCD formation with continuous extra expression of Hp by gene therapy in hepatic tissue. Although previous studies did not observe good transgene expression in hepatic tissue using cationic liposome solution inoculated via the tail vein of rats [28], it appears that the orbital plexus route was effective in our case. Contrary to the results obtained by those authors, we did not observe transgene expression in the lung and spleen, but rather effective expression in hepatic tissue. These results indicate that efficient plasmid delivery and marker expression were achieved, establishing a solid methodological foundation for our objectives.

Our results obtained in this study partially corroborate the literature, demonstrating beneficial effects associated with Hp. Although its action is primarily described as occurring after hemolysis, improvements in hemolytic status and increased erythrocyte resistance were observed, possibly through indirect mechanisms related to the reduction of the toxic effects of free hemoglobin. Interestingly, haptoglobin acts after hemolysis by clearing free hemoglobin from the organism; however, our results indicate an improvement in the hemolytic condition and increased erythrocyte resistance. To date, there is no evidence of a direct effect of haptoglobin in reducing hemolysis, and the observed results may be due to an indirect action—through the reduction of the harmful effects of free hemoglobin in an organism with chronic hemolysis. Our findings are consistent with a study that reported very similar results to ours, showing improvements in organ anatomy as well as hematological parameters in beta-thalassemic mice treated with an apohemoglobin–haptoglobin complex [25]. Like them, we believe that the reduction or control of the release of harmful factors from hemolysis into the bloodstream, even in organisms with chronic hemolysis—such as in sickle cell and thalassemic conditions—can lead to a decrease in factors like cytokines and enzymes that worsen the inflammatory process and oxidative stress. This may favor physiological hemolysis, rather than being solely attributed to the genetic deficiency of red blood cells. Although we did not conduct in-depth studies on inflammation, our results may suggest this theory. Another study also in thalassemia demonstrated the slowing of arteriosclerosis as an indirect effect of hemopexin treatment [13]. In addition to these observations, the extended hematological analysis provided further insight into the effects of haptoglobin treatment. Treated animals exhibited increased red blood cell (RBC) counts, reinforcing the observed improvements in hematocrit and hemoglobin levels and supporting enhanced erythrocyte survival and/or reduced hemolysis. Platelet counts were also significantly elevated, suggesting a partial recovery of hematopoietic function and a possible reduction of inflammatory process, although this finding should be interpreted with caution given the complex role of platelets in SCD pathophysiology.

In contrast, no significant changes were observed in total leukocyte counts, which showed considerable variability among samples. Differential leukocyte analysis, including neutrophil counts, was not performed, limiting a more detailed evaluation of inflammatory responses. Similarly, %HbS levels remained unchanged between groups, indicating that the therapeutic effects of haptoglobin are likely associated with modulation of hemolysis and its downstream consequences rather than direct alterations in hemoglobin composition.

In line with these observations, despite our study could not precisely determine the anti-hemolytic effect mediated by haptoglobin overexpression, the work by Hurtado et al. (2023) suggests indirect beneficial effects on the attenuation of inflammatory and vascular processes in experimental models of hemolytic diseases treated with hemopexin [26]. A more comprehensive analysis of inflammatory markers would be required to better elucidate the underlying mechanism.

Overall, the plasmid construct containing the human Hp gene demonstrated efficient in vivo expression and promoted relevant therapeutic effects in the SCD model, including hematological improvement, reduction of compensatory erythropoiesis, and attenuation of clinical signs such as splenomegaly. Partial preservation of organ structure was also observed, particularly in the liver and spleen, although without complete reversal of microvascular alterations, reinforcing the potential of recombinant Hp as a therapeutic strategy and highlighting the need for further optimization of this approach.

Histological analysis in murine models proved to be essential for understanding the cellular and tissue-level effects of the interventions, allowing the identification of structural, inflammatory, and regenerative changes. In SCD, marked alterations are observed mainly in the spleen, liver, and kidneys. In the spleen, autosplenectomy may occur as a result of repeated infarctions caused by vascular obstruction, leading to fibrosis, atrophy, and impaired immune function [29], whereas in healthy conditions the splenic architecture remains preserved, with proper organization of the white and red pulp [30]. In the liver, sinusoidal dilation and inflammatory infiltration associated with chronic hemolysis are observed, while in the kidneys there is inflammation, edema, and microvascular congestion, compromising renal function [31]. These findings reinforce the systemic effects of SCD and highlight the impact of continuous hemolysis on multiple organs.

A pioneering study evaluating repeated administration of synthetic haptoglobin (Hp) in a murine model of sickle cell disease (Townes mice) demonstrated modest therapeutic effects on disease-associated organ damage [32]. Administration of high doses of synthetic Hp (400 mg/kg) increased the expression of heme oxygenase-1 (HO-1) and ferritin heavy chain (ferritin-H), while reducing iron deposition in the kidneys. In addition, a trend toward a reduced incidence of hepatic infarction was observed. Although this latter finding is consistent with our observations, the remaining outcomes differ substantially, as the therapeutic strategy employed in the present study produced broader and more pronounced biological effects.

In our experimental model, sustained Hp overexpression induced by gene therapy resulted in significant histological improvement of the spleen, together with a consistent recovery of erythroid hematological parameters, as evidenced by the attenuation of anemia and the overall improvement of the hematological indices previously described and discussed. These findings suggest that continuous Hp expression exerted protective effects not only against tissue injury but also on the hematological pathophysiology that characterizes sickle cell disease.

The differences observed between the two studies are likely attributable to differences in both the experimental model and the therapeutic strategy employed. Whereas the previous study was based on the intermittent administration of synthetic Hp in Townes mice, our model was established by transplantation of bone marrow from Berkeley mice with severe sickle cell disease into lethally irradiated C57BL/6 recipient mice, generating a stable and severe hematological phenotype approximately 60 days after transplantation. Therapeutic intervention was initiated at this stage, when the pathological process was already established and the initial manifestations of organ injury were developing.

In this context, sustained Hp expression appears to have primarily delayed or attenuated the progression of hemolysis-associated comorbidities rather than completely preventing their development. These findings reinforce the importance of modulating the Hp-free hemoglobin (Hb) scavenging pathway to mitigate the progressive tissue damage associated with sickle cell disease, while also indicating that this strategy alone is not sufficient to fully halt disease progression.

The efficacy of this mechanism is likely dependent on the balance between the amount of available Hp and the continuous burden of free hemoglobin released during chronic intravascular hemolysis. In sickle cell disease, endogenous Hp production rapidly becomes insufficient to neutralize the high concentrations of extracellular Hb, resulting in saturation of the physiological Hp-Hb scavenging system. From this perspective, gene therapy approaches capable of promoting sustained, high-level Hp expression may represent a promising therapeutic strategy, particularly when combined with the initial administration of exogenous recombinant Hp. Such a combined approach would enable the rapid neutralization of circulating free Hb present at the onset of treatment, while continuous Hp expression driven by gene therapy would progressively restore the capacity for extracellular Hb clearance, thereby re-establishing vascular homeostasis and reducing the deleterious consequences of chronic hemolysis.

The interpretation of the present findings should consider experimental limitations inherent to studies involving severe sickle cell disease (SCD) animal models. The generation and maintenance of these models are technically demanding due to their low reproductive performance, high susceptibility to experimental manipulation, and reduced survival, all of which substantially limit sample availability. These challenges are further compounded in bone marrow transplantation-based models, in which achieving homogeneous levels of sickle hemoglobin (HbS) expression among recipient animals represents an additional source of biological variability.

To minimize this variability, only animals exhibiting the highest degree of similarity in HbS levels were included in the experimental groups, thereby increasing the consistency and internal validity of the analyses despite the relatively small sample size. Although this approach inevitably reduced the number of animals available for statistical analysis, it strengthened the biological comparability between groups and increased the reliability of the observed treatment effects.

Another important limitation was the restricted volume of blood that could be collected from animals presenting with severe anemia, which constrained the number of complementary analyses that could be performed. Consequently, additional investigations, including comprehensive inflammatory profiling, longitudinal quantification of circulating cell-free hemoglobin before and after treatment, and pharmacokinetic assessment of circulating Hp concentrations throughout the treatment period, could not be performed. Such analyses would provide valuable mechanistic insights into the protective effects observed in the present study.

The continuous intravascular hemolysis characteristic of SCD, together with the rapid formation and subsequent clearance of Hp-Hb complexes, likely resulted in rapid consumption of the administered Hp, leaving circulating concentrations below the detection limit of the analytical method employed. Therefore, more sensitive analytical approaches will be required in future studies to accurately characterize Hp pharmacokinetics and to establish the relationship between circulating Hp levels, free Hb scavenging capacity, and therapeutic efficacy. Such investigations will be essential for optimizing Hp-based therapeutic strategies and further elucidating the mechanisms underlying the beneficial effects observed in the present study.

Furthermore, the results obtained open perspectives for future investigations, including the improvement of haptoglobin-based therapeutic approaches, the development of combined strategies, and the expansion of diagnostic and therapeutic applications in hemolytic diseases, reinforcing the importance of continuing this line of research.

Data Availability

DOI: https://doi.org/10.25824/redu/KYLRCN.

Funding Statement

São Paulo Research Foundation (FAPESP) for supporting project nº 2019/18886-1, and the Coordination for the Improvement of Higher Education Personnel (CAPES) project nº 88887.676144/2022-00, linked to the Ministry of Education of Brazil. There was no additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0357180.r001

Decision Letter 0

Fábio Silva

13 Oct 2025

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Additional Editor Comments:

Dear Dr. Corat,

Thank you for submitting your manuscript entitled “Therapeutic potential of haptoglobin in a murine model of sickle cell anemia” to PLOS ONE.

The study addresses a relevant and timely topic and has potential translational impact. However, one of the reviewers raised several major methodological and reporting concerns that must be addressed before the manuscript can be reconsidered for publication.

Key points to address in your revision include:

1. Report the number of animals used in each group (total transplanted, excluded, final N), and present individual data points in the figures rather than only bar graphs.

2. Provide a justification for the choice of haptoglobin 1, and discuss the clinical implications of this decision in the context of Hp1 versus Hp2.

3. Describe in detail how and when SS bone marrow engraftment was confirmed, and discuss the potential impact of circulating wild-type red blood cells at the time of treatment.

4. Address whether inflammatory parameters (e.g., leukocytes, neutrophils) were evaluated after treatment, and discuss their relevance to the therapeutic potential of haptoglobin.

5. Add missing references, clearly state the model used, anesthetic protocols, and software employed for analysis.

6. Expand the Discussion section. We recommend separating “Results” and “Discussion” into two distinct sections to improve clarity, structure, and scientific depth. The discussion should compare and contrast your findings with relevant previous studies that have investigated haptoglobin treatment in SCD models, highlighting advances and limitations.

7. Revise the textual issues and figure labeling inconsistencies noted by the reviewer (e.g., figure numbering, references, terminology).

8. Given the significance of these points, the editorial decision is Major Revision. Please submit a revised version of your manuscript with a detailed, point-by-point response to each reviewer comment.

We look forward to receiving your revised manuscript.

Sincerely,

Dr. Fabio Henrique Silva

Academic Editor

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[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: N/A

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: General questions:

1-What is the number of mice used? At no point did I see how many mice were transplanted, how many had the SS bone marrow implanted, or how many were discarded. The N of mice in the figures are also not included. Furthermore, the graphs could be plotted as points for each number, rather than a closed bar.

2-Why did you choose overexpression of haptoglobin 1 and not 2? Or both for have the same human phenotype.

3- Knowing that mouse red blood cells have a lifespan of 40-60 days, healthy red blood cells continue to circulate for this period after transplantation. Did you confirm SS marrow engraftment by hemoglobin electrophoresis/liquid chromatography? Or what method?

In line 196 it looks like to have been done by chromatography, but it is not clear how this sample was collected and at what point post-transplant.

Furthermore, in line 181, you said that the treatment was carried out 1 week after the transplant, so you treated the animals without knowing if the SS marrow had engaged, and even if it had switched to SS, the 'wild' red blood cells continued to circulate until approximately the date of sacrifice, which was 60 days after the treatment. Would this be a limitation of study?

4- Considering Haptoglobin as a possible treatment for sickle cells, after your treatment, did you see any decrease in inflammatory parameters? such as leukocytes and especially neutrophils? In the blood count?

__________

Line 24 - where you say -the genetic, you can remove it, continue- with sickle cell disease...

Line 53 – Need add reference.

Line 59 - A reference from 1969 was cited in the text, in the references look like a thesis from 2008, please cite the original reference.

Line 65 - blood components, such as renal cells, if they are renal cells are not blood components.

The text from line 62 to 71 needs to be better referenced.

Line 76 – Need add reference.

Line 94 - Although you included the genetic variety, they didn't include the model’s name. Are these MO donor animals Tim Townes or Berkeley? This makes it easier for the reader.

Line 95 - Include only the first author, do not include all of them.

Line 102- Include the concentration and/or % and proportion of the anesthetics.

Line 108 - What species are these mice that were irradiated and received the bone marrow?

Line 173 - which software?

Lines 239 to 276 – only described the results and did not discuss them with previous work. I think the entire work needs to be better discussed; most of the results and discussion sections are just describing the results and not discussing them.

Line 287-296-297 -why did you choose 26 days? And not before or after? Also, including Figure 3, do you have an image of the PBS control SS? I think it would be important to show the difference in images.

Line 301- Fig 3 when in fact it is figure 4.

Line 312 - Reference normal range.

Line 313- Suggests RBC production, why was the number of RBCs not included in Figure 4?

Line 320 - Did you try to measure free hemoglobin? Additionally, did the plasma haptoglobin levels change?

Reviewer #2: The study is relevant and presents a promising gene therapy strategy using haptoglobin in a murine model of sickle cell anemia. The data are consistent with the conclusions, and the work makes a significant contribution to the field of experimental therapies.

Some suggestions are to provide the raw data (individual values) and to clearly report the number of animals (n) in each experimental group.

**********

what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Tammyris Helena Rebecchi e Silveira

**********

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PLoS One. 2026 Sep 18;21(9):e0357180. doi: 10.1371/journal.pone.0357180.r002

Author response to Decision Letter 1

27 May 2026

We would like to thank the reviewers for their careful evaluation of our manuscript, as well as for their valuable comments and suggestions. We sincerely appreciate the time and effort dedicated to the assessment of our work.

All comments were carefully considered, and the manuscript has been revised accordingly. The changes made are highlighted in the revised version, and detailed responses to each point raised are provided below.

We believe that these revisions have significantly improved the clarity and overall quality of the manuscript.

________________________________________

Editor’s Comment:

Please include the following items when submitting your revised manuscript: a response letter, a marked-up version of the manuscript, and a clean version of the manuscript.

Authors’ Response:

All requested documents have been properly prepared and submitted according to the instructions: (i) the response letter to the reviewers, (ii) the revised manuscript with tracked changes, and (iii) the final clean version of the manuscript without markings.

Editor’s Comment:

If you wish to make changes to your financial disclosure, please include the updated version in your cover letter. We also recommend depositing your laboratory protocols in protocols.io.

Authors’ Response:

The cover letter has been reviewed and updated as necessary. Regarding the recommendation to make protocols/data available, we inform that these have been deposited in the institutional repository of the University of Campinas (REDU), with the corresponding identifier: https://doi.org/10.25824/redu/KYLRCN

Editor’s Comment:

Please ensure that your manuscript meets the PLOS ONE style requirements, including file naming conventions.

Authors’ Response:

The manuscript has been reviewed and adjusted to comply with PLOS ONE style requirements, including formatting and file naming, as instructed.

Editor’s Comment:

Provide additional information in the Methods section regarding animal experiments, including euthanasia methods, anesthesia/analgesia, and efforts to alleviate suffering.

Author Response:

We thank the reviewer for this comment. The requested information has been included and detailed in the Methods section of the revised manuscript.

Briefly, animals were anesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg), ensuring adequate sedation, analgesia, and muscle relaxation for the experimental procedures. For euthanasia, a high anesthetic dose (300 mg/kg ketamine and 30 mg/kg xylazine) was administered, followed by cervical dislocation, in accordance with the guidelines of the Animal Ethics Committee (CEUA) and CONCEA.

In addition, measures were implemented throughout the entire experiment to minimize animal suffering, including continuous monitoring, appropriate anesthesia and analgesia protocols, and procedures conducted by a trained team, in compliance with the 3Rs principles.

Editor’s Comment:

A revised Funding Statement is requested, including all sources of support, as well as the inclusion of the sentence “No additional external funding was received for this study.”

Author Response:

We thank the reviewer for this comment. The Funding Statement has been revised and updated to include all sources of support received during the conduct of the study, in accordance with the journal guidelines. The requested sentence (“No additional external funding was received for this study”) has also been included. The updated Funding Statement has been incorporated into the cover letter.

Editor’s Comment:

Please indicate the role of the funders in the study, including the appropriate statement in the cover letter.

Author Response:

We thank the reviewer for this comment. The statement regarding the role of the funders has been included in the cover letter, as requested. Specifically, we state that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Editor’s Comment:

Please remove funding information from the Acknowledgements section and update the Funding Statement.

Author Response:

We thank the reviewer for this comment. The funding-related information has been removed from the Acknowledgements section of the manuscript, as instructed.

The Funding Statement has been revised and updated in accordance with the journal guidelines, and the corrected version has been included in the cover letter for the corresponding updates in the submission system.

Editor’s Comment:

Please confirm and, if necessary, update the Data Availability Statement, ensuring access to the minimum dataset required to replicate the study.

Author Response:

We thank the reviewer for this comment. The Data Availability Statement has been revised to ensure compliance with the PLOS ONE guidelines.

The minimum dataset required for replication of the study results has been made publicly available in an institutional repository (REDU – University of Campinas), with the corresponding link/DOI: https://doi.org/10.25824/redu/KYLRCN

In addition, we confirm that all data relevant to the interpretation of the results are included within the manuscript, the supplementary files, and/or the indicated repository.

Editor’s Comment:

Figure 3 contains copyrighted images. Please obtain permission or replace the figure with content compliant with the CC BY 4.0 license.

Author Response:

We thank the reviewer for this comment. Figure 3 has been replaced with a new original version, without the use of any previously copyrighted material, and is fully compliant with the CC BY 4.0 license requirements.

Reviewer Comment:

What is the number of mice used? [...] In addition, the graphs could be presented as individual points instead of bar graphs.

Author Response:

We thank the reviewer for this comment. Information regarding the number of animals has been included and detailed in the revised manuscript.

Six animals were transplanted in each experimental group. In both groups, deaths occurred, with one death recorded in the treated group and two in the control group. In the treated group, one animal showed a high level of chimerism (~95%), while another presented a very low level and was therefore excluded from the analysis. Thus, three animals with intermediate levels of chimerism (mean approximately 75%) were included in the analysis.

In the control group, animals with chimerism levels comparable to those observed in the treated group were selected, totaling three animals for analysis.

Additionally, the number of animals (n = 3) has been included in the figure legends. We consider that this information allows appropriate interpretation of the presented results.

Reviewer Comment:

Why was haptoglobin 1 overexpression chosen instead of haptoglobin 2? Please justify this choice and discuss the clinical implications in the context of Hp1 versus Hp2 comparison.

Author Response:

We thank the reviewer for this comment. The choice of haptoglobin phenotype Hp1 was based on literature evidence demonstrating the greater efficiency of this phenotype in neutralizing the deleterious effects of free hemoglobin.

Studies indicate that Hp1-1 exhibits higher antioxidant capacity compared to Hp2-2, promoting the formation of more stable Hb-Hp complexes with more efficient clearance via endocytosis. In contrast, complexes formed with Hp2-2 are associated with increased generation of reactive oxygen species, thereby enhancing the potential for oxidative damage to lipids, proteins, and DNA.

Therefore, considering the context of chronic hemolysis observed in sickle cell anemia, Hp1 was considered more appropriate as a therapeutic strategy due to its greater efficiency in reducing oxidative stress and its potential tissue-protective effects (LEVY et al., 2010; PIRES; BIERHALZ; MORAES, 2015; PIRES; BERTHIAUME; PALMER, 2023; QUIMBY; HAMBLETON; LANDIS, 2015).

The clinical implications of this choice have been included and further discussed in the revised version of the manuscript.

Reviewer Comment:

Describe how and when the SS bone marrow graft was confirmed, which method was used, and discuss the impact of circulating wild-type red blood cells at the time of treatment.

Author Response:

We thank the reviewer for this comment. The engraftment was confirmed by high-performance liquid chromatography (HPLC) using the Variant II automated system (Bio-Rad). Peripheral blood samples were collected via submandibular puncture (cheek pouch), subsequently aspirated, injected into the analytical system, and applied to a cation-exchange column. A programmed buffer gradient enabled the separation of different hemoglobin fractions based on their ionic interactions with the column. Eluted fractions were detected by photometry at 415 nm, identified by their characteristic retention times, and quantified by peak area integration.

The transplanted models were considered to exhibit a severe sickle cell anemia phenotype 60 days post-transplantation, with an HbS proportion above 70%, as described by Iannone et al. (2001).

Regarding the presence of wild-type red blood cells, it is important to note that gamma-irradiated erythrocytes exhibit a significantly reduced lifespan, estimated at approximately 10 days, as reported in the literature. Therefore, at 60 days post-transplantation, animals predominantly present erythrocytes derived from the transplanted bone marrow, expressing physiological and molecular features consistent with sickle cell anemia. Additionally, in the absence of transplantation, animals do not survive beyond 7–10 days due to the lack of functional erythrocytes.

These points have been included and discussed in the revised manuscript.

Reviewer Comment:

Indicate whether inflammatory parameters (e.g., leukocytes, neutrophils) were assessed after treatment and discuss their relevance to the therapeutic potential of haptoglobin.

Author Response:

We thank the reviewer for this comment. Differential leukocyte counts were not performed in the present study. Regarding total leukocyte counts, high variability among samples was observed, and no statistically significant differences were identified between groups.

On the other hand, a significant increase in platelet counts was observed in the treated animals.

We emphasize that a more precise evaluation of the inflammatory profile would require the assessment of specific markers, such as cytokines, which were not performed in this study; however, this approach will be considered in future investigations.

Nevertheless, as described in the literature (Hurtado et al., 2023), hemolytic conditions are associated with increased levels of free heme, which can promote oxidative stress and inflammatory responses through activation of innate immune pathways. Therefore, by reducing circulating free hemoglobin and its downstream heme-related toxicity, haptoglobin may indirectly contribute to the modulation of inflammatory processes.

Reviewer Comment:

Minor corrections in the text, including conceptual adjustments, addition of references, citation standardization, methodological detailing, and figure corrections (lines 24, 53, 59, 62–71, 65, 76, 95, 102, 173, 301, 312, and 313).

Author Response:

We thank the reviewer for these comments. All minor corrections have been addressed in the revised manuscript, including the removal and adjustment of terms, addition and correction of references, standardization of citations, further methodological detailing, and correction of inconsistencies in both the figures and the text.

In addition, the requested information, such as reference ranges and the mentioned hematological parameters, has been included.

Reviewer Comment:

Although genetic variability was included, the model name was not mentioned. Are the bone marrow donor animals from the Townes or Berkeley model?

Author Response:

We thank the reviewer for this comment. The bone marrow donor mice used in this study belong to the Berkeley model, a widely used humanized model of sickle cell disease that predominantly expresses human hemoglobin S.

Additionally, the irradiated recipient animals belong to the species Mus musculus, C57BL/6J strain.

This information has been included in the revised manuscript.

Reviewer Comment:

The manuscript presents several issues related to the discussion of the results, which is predominantly descriptive. It is recommended to expand the discussion by incorporating critical analysis and comparison with previous studies in the literature.

Author Response:

The Discussion section has been revised and expanded, including comparisons with previous studies in the literature, in order to provide a more in-depth critical analysis of the obtained results.

Reviewer Comment:

Why was the 26-day time point chosen for analysis? In addition, please include an image of the PBS SS control in Figure 3.

Author Response:

We thank the reviewer for this comment. The 26-day time point for the analyses was selected based on logistical aspects related to experimental organization and equipment availability. Nevertheless, this period proved to be appropriate for the manifestation of the evaluated changes, as demonstrated by the results obtained in the study.

Additionally, as suggested, the image corresponding to the PBS SS control was included in the revised figure, allowing for a better comparison between the experimental groups.

Reviewer Comment:

Erythrocyte production is suggested; why was the number of erythrocytes not included in Figure 4?

Author Response:

We thank the reviewer for this comment. The erythrocyte count has been included in the revised version of Figure 4, as suggested.

Reviewer Comment:

Did you attempt to measure free hemoglobin? In addition, did plasma haptoglobin levels change?

Author Response:

We thank the reviewer for this comment. The amount of biological material available (blood) was limited, which restricted the performance of additional analyses, including the measurement of free hemoglobin.

Regarding plasma haptoglobin, attempts were made to quantify it; however, the values obtained did not reach the minimum detection limit of the equipment used. Considering the context of sickle cell anemia, in which there is marked consumption of haptoglobin due to its binding to free hemoglobin, a detectable level of this parameter was not expected.

Therefore, it was not possible to obtain reliable measurements for these markers in the present study.

________________________________________

Closing Remarks

We would like to thank the Editor and the reviewers once again for their careful evaluation of the manuscript and for the valuable suggestions provided. All comments were carefully considered, and we believe that the revisions made have significantly improved the clarity, consistency, and scientific quality of the work.

We hope that the revised version meets the expectations, and we remain available for any further clarifications that may be required.

Attachment

Submitted filename: Response to reviewers.docx

pone.0357180.s002.docx^{(1.6MB, docx)}

PLoS One. doi: 10.1371/journal.pone.0357180.r003

Decision Letter 1

Fábio Silva

5 Jun 2026

Dear Dr. Corat,

Please submit your revised manuscript by Jul 20 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Fábio Henrique Silva, Ph.D.

Academic Editor

PLOS One

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise.

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments :

Dear Authors,

Thank you for submitting the revised version of the manuscript. The revision has improved the clarity of the study, particularly regarding the animal model, HPLC analysis, experimental groups, and rationale for using haptoglobin 1. However, a few points still need to be addressed before the manuscript can be considered for acceptance.

1. Revise graphical presentation of quantitative data

The previous reviewer suggested that graphs should display individual data points rather than only closed bars. In the current version, Figure 4 still appears to be presented as bar graphs with mean ± SD. Please revise Figure 4 to show individual data points for each animal, with mean ± SD or median and dispersion as appropriate. Given the final n = 3 per group, showing individual values is essential for transparent interpretation.

2. Add a final limitations paragraph

Please add a dedicated limitations paragraph at the end of the Discussion. This paragraph should explicitly address the main limitations of the study, including the small final sample size, the absence of direct quantification of circulating free hemoglobin and free heme, the lack of reliable plasma haptoglobin measurement, and the limited inflammatory assessment. In particular, the manuscript currently discusses possible anti-inflammatory effects. However, total WBC counts did not differ significantly between groups, differential leukocyte counts were not performed, and neutrophils, cytokines, or tissue inflammatory markers were not quantified. Therefore, conclusions regarding inflammation should be interpreted cautiously and primarily based on histopathological observations.

3. Contextualize the findings with previous haptoglobin-based studies in SCD models

Please briefly discuss previous evidence from sustained haptoglobin treatment in sickle cell mice, particularly the study “Sustained treatment of sickle cell mice with haptoglobin increases HO-1 and H-ferritin expression and decreases iron deposition in the kidney without improvement in kidney function” published in British Journal of Haematology in 2016.

This study is relevant because haptoglobin treatment increased markers related to heme and iron handling, including HO-1 and H-ferritin, and reduced renal iron deposition, but did not improve hematological parameters or kidney function. Since the present manuscript reports improvement in hematological parameters after haptoglobin overexpression, the authors should briefly discuss how their findings compare with this previous study.

The discussion should address whether differences between the studies may be related to the therapeutic strategy used, gene-based haptoglobin overexpression versus sustained haptoglobin protein treatment, treatment duration, disease model, timing of analysis, or sample size. This comparison will help readers interpret whether the hematological improvement observed in the present study reflects a distinct effect of the experimental approach or should be considered exploratory and requiring further confirmation.

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PLoS One. 2026 Sep 18;21(9):e0357180. doi: 10.1371/journal.pone.0357180.r004

Author response to Decision Letter 2

19 Jul 2026

We believe that these revisions have significantly improved the clarity and overall quality of the manuscript.

________________________________________

Editor’s Comment:

Please include the following items when submitting your revised manuscript: a response letter, a marked-up version of the manuscript, and a clean version of the manuscript.

Authors’ Response:

Editor’s Comment:

If you wish to make changes to your financial disclosure, please include the updated version in your cover letter. We also recommend depositing your laboratory protocols in protocols.io.

Authors’ Response:

Editor’s Comment:

Please ensure that your manuscript meets the PLOS ONE style requirements, including file naming conventions.

Authors’ Response:

The manuscript has been reviewed and adjusted to comply with PLOS ONE style requirements, including formatting and file naming, as instructed.

Editor’s Comment:

Provide additional information in the Methods section regarding animal experiments, including euthanasia methods, anesthesia/analgesia, and efforts to alleviate suffering.

Author Response:

We thank the reviewer for this comment. The requested information has been included and detailed in the Methods section of the revised manuscript.

Editor’s Comment:

A revised Funding Statement is requested, including all sources of support, as well as the inclusion of the sentence “No additional external funding was received for this study.”

Author Response:

Editor’s Comment:

Please indicate the role of the funders in the study, including the appropriate statement in the cover letter.

Author Response:

Editor’s Comment:

Please remove funding information from the Acknowledgements section and update the Funding Statement.

Author Response:

We thank the reviewer for this comment. The funding-related information has been removed from the Acknowledgements section of the manuscript, as instructed.

Editor’s Comment:

Please confirm and, if necessary, update the Data Availability Statement, ensuring access to the minimum dataset required to replicate the study.

Author Response:

We thank the reviewer for this comment. The Data Availability Statement has been revised to ensure compliance with the PLOS ONE guidelines.

In addition, we confirm that all data relevant to the interpretation of the results are included within the manuscript, the supplementary files, and/or the indicated repository.

Editor’s Comment:

Figure 3 contains copyrighted images. Please obtain permission or replace the figure with content compliant with the CC BY 4.0 license.

Author Response:

Editor’s Comment:

Revise graphical presentation of quantitative data. The previous reviewer suggested that graphs should display individual data points rather than only closed bars. In the current version, Figure 4 still appears to be presented as bar graphs with mean ± SD. Please revise Figure 4 to show individual data points for each animal, with mean ± SD or median and dispersion as appropriate. Given the final n = 3 per group, showing individual values is essential for transparent interpretation.

Author Response:

We thank the editor for this comment. Figure 4 has been replaced with a new original version as the reviewer suggestion.

Editor’s Comment:

Author Response:

We thank the editor for this comment. The limitations paragraphs have been added.

Lines 568-602. In resume: the interpretation of the present findings should consider the inherent limitations of severe sickle cell disease (SCD) animal models, including their low reproductive performance, high susceptibility to experimental manipulation, reduced survival, and the biological variability associated with bone marrow transplantation-based models. To minimize this variability, only animals with comparable sickle hemoglobin (HbS) levels were included, improving the internal consistency of the study despite the limited sample size.

An additional limitation was the small volume of blood that could be collected from severely anemic animals, restricting complementary analyses such as inflammatory profiling, longitudinal assessment of circulating cell-free hemoglobin, and pharmacokinetic evaluation of haptoglobin (Hp). Furthermore, circulating Hp concentrations could not be reliably quantified, likely due to the rapid consumption of Hp following complex formation with free hemoglobin during chronic intravascular hemolysis, resulting in concentrations below the detection limit of the analytical method. Therefore, future studies employing more sensitive analytical techniques will be necessary to characterize Hp pharmacokinetics and further elucidate the relationship between circulating Hp levels, free Hb scavenging, and therapeutic efficacy.

Editor’s Comment:

Contextualize the findings with previous haptoglobin-based studies in SCD models

Author Response:

We thank the editor for this comment. Paragraphs contextualizing previous studies have been added.

Lines 526-567. In General, a previous study evaluating repeated administration of synthetic haptoglobin (Hp) in Townes sickle cell mice reported modest therapeutic effects, including increased HO-1 and ferritin-H expression, reduced renal iron deposition, and a trend toward decreased hepatic infarction (32). In contrast, our gene therapy approach induced sustained Hp expression and produced broader therapeutic benefits, including significant improvement in splenic histology and erythroid hematological parameters.

These differences are likely attributable to the distinct experimental models and treatment strategies. While continuous Hp expression attenuated the progression of hemolysis-associated tissue damage, it did not completely prevent disease progression, indicating that Hp supplementation alone is insufficient to fully restore homeostasis.

Given the continuous release of free hemoglobin during chronic hemolysis, combining sustained Hp expression through gene therapy with the initial administration of exogenous recombinant Hp may represent a more effective therapeutic strategy by providing both immediate and long-term extracellular hemoglobin scavenging.

Reviewer Comment:

What is the number of mice used? [...] In addition, the graphs could be presented as individual points instead of bar graphs.

Author Response:

We thank the reviewer for this comment. Information regarding the number of animals has been included and detailed in the revised manuscript.

In the control group, animals with chimerism levels comparable to those observed in the treated group were selected, totaling three animals for analysis.

Additionally, the number of animals (n = 3) has been included in the figure legends. We consider that this information allows appropriate interpretation of the presented results.

Reviewer Comment:

Why was haptoglobin 1 overexpression chosen instead of haptoglobin 2? Please justify this choice and discuss the clinical implications in the context of Hp1 versus Hp2 comparison.

Author Response:

The clinical implications of this choice have been included and further discussed in the revised version of the manuscript.

Reviewer Comment:

Describe how and when the SS bone marrow graft was confirmed, which method was used, and discuss the impact of circulating wild-type red blood cells at the time of treatment.

Author Response:

The transplanted models were considered to exhibit a severe sickle cell anemia phenotype 60 days post-transplantation, with an HbS proportion above 70%, as described by Iannone et al. (2001).

These points have been included and discussed in the revised manuscript.

Reviewer Comment:

Indicate whether inflammatory parameters (e.g., leukocytes, neutrophils) were assessed after treatment and discuss their relevance to the therapeutic potential of haptoglobin.

Author Response:

On the other hand, a significant increase in platelet counts was observed in the treated animals.

Nevertheless, as described in the literature (Hurtado et al., 2023), hemolytic conditions are associated with increased levels of free heme, which can promote oxidative stre

Attachment

Submitted filename: Response_to_reviewers_auresp_2.docx

pone.0357180.s003.docx^{(1.6MB, docx)}

PLoS One. doi: 10.1371/journal.pone.0357180.r005

Decision Letter 2

Fábio Silva

13 Aug 2026

Therapeutic potential of haptoglobin in a murine model of sickle cell anemia

PONE-D-25-51689R2

Dear Dr. Corat,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Fábio Henrique Silva, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0357180.r006

Acceptance letter

Fábio Silva

PONE-D-25-51689R2

PLOS One

Dear Dr. Corat,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team.

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PLOS One

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Attachment

Submitted filename: Response to reviewers.docx

pone.0357180.s002.docx^{(1.6MB, docx)}

Attachment

Submitted filename: Response_to_reviewers_auresp_2.docx

pone.0357180.s003.docx^{(1.6MB, docx)}

Data Availability Statement

DOI: https://doi.org/10.25824/redu/KYLRCN.

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Therapeutic potential of haptoglobin in a murine model of sickle cell anemia

Bianca Cristina dos Santos

Magnun Nueldo Nunes dos Santos

Gisele Audrei Pedroso

Beatriz Benedetti de Oliveira

Dulcinéia Martins de Albuquerque

Nathalia Rocha de Oliveira

Daniela Cagnoto Noronha

Fernando Ferreira Costa

Marcus Alexandre Finzi Corat

Roles

Abstract

Introduction

Materials and methods

Bone marrow transplantation to generate SCD mouse models

Haptoglobin cloning using the pGEM-T Easy vector

Cloning of haptoglobin into the Lego-tdTomato plasmid

Sequencing

Introduction of the plasmid into animals

Pathological analysis

Results

Fig 1. Schematic representation of haptoglobin expression vector construction and validation.

Fig 2. Sequencing analysis of the cloned haptoglobin gene.

Fig 3. Plasmid delivery and hepatic expression in SCD mice (n = 3).

Fig 4. Hematological parameters and spleen size in SCD mice after haptoglobin plasmid treatment (n = 3).

Fig 5. Histological changes in kidney, spleen, and liver of SCD mice after haptoglobin plasmid treatment (n = 3).

Discussion

Data Availability

Funding Statement

References

Decision Letter 0

Fábio Silva

Roles

Author response to Decision Letter 1

Decision Letter 1

Fábio Silva

Roles

Author response to Decision Letter 2

Decision Letter 2

Fábio Silva

Roles

Acceptance letter

Fábio Silva

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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