Molecular mechanisms of hepatic dysfunction in sickle cell disease: lessons from Townes mouse model

Abstract

Sickle cell disease (SCD) is an autosomal recessive genetic disorder that affects ∼100,000 Americans and millions of people worldwide. Erythrocyte sickling, vaso-occlusion, sterile inflammation, and hemolysis are the major pathophysiological pathways leading to liver injury in SCD. Although hepatic dysfunction affects up to 10%–40% of patients with SCD, therapeutic approaches to prevent liver injury in SCD are not known, and the molecular mechanisms promoting progressive liver injury in SCD remain poorly understood. Animal models have been beneficial in bridging the gap between preclinical and translational research in SCD. Recent advances in methodology have allowed the development of several humanized animal models to address various aspects of SCD-related liver diseases. This review provides an overview of current knowledge of the molecular mechanisms and potential therapeutic options of SCD-associated liver dysfunction using the Townes mouse model.

INTRODUCTION

Sickle cell disease (SCD) is an autosomal recessive monogenic disorder that affects millions of people worldwide, with an estimated annual medical cost of over $1.1 billion in the United States (1–5). A point mutation at the sixth position in the β-globin gene substituting glutamic acid with valine results in sickled hemoglobin (HbS) (4). Patients homozygous for this mutation show vaso-occlusion, intense hemolysis, and sterile inflammation (Fig. 1), which promote chronic damage to multiple organs (3, 4, 6, 7).

Figure 1.

How is the liver affected in SCD? Schematic showing the different pathophysiology associated with SCD that can cause hepatic dysfunctions. SCD induced hemolysis, vaso-occlusion, endothelial cell activation, increase of DAMPs and PAMPs, exacerbated inflammation, and hepatic iron accumulation that can each lead to acute and chronic liver injury as seen in Townes SCD mice model. DAMP, damage-associated molecular pattern; PAMP, pathogen-associated molecular pattern; SCD, sickle cell disease.

According to a systematic analysis of the Global Burden of Disease study, 3.2 million people have SCD, 43 million people are carriers of the SCD trait, and 176,000 people die each year from SCD-related complications (8). Patients with SCD have a reduced life expectancy (1). However, as a result of recent breakthroughs in therapy and increased quality of life, patients with SCD live longer, which contributes to an increase in the occurrence of organ damage (9). Although the disease proceeds with pain episodes (5), persistent organ damage is often silent until the condition is severe (10). The common chronic complications described in adult patients with SCD include pulmonary hypertension (7, 11–13), kidney disease (14, 15), avascular necrosis (16), retinopathy (17), acute chest syndrome (18), and sickle hepatopathy (19–22).

Spectrum of Acute and Chronic Liver Problems Seen in Patients with SCD

“Sickle hepatopathy” refers to a range of liver symptoms in people living with SCD, from mild asymptomatic liver function test abnormalities to acute liver failure phenotypes such as severe hyperbilirubinemia to cirrhosis with liver failure (20, 23–25). A total of 10%–40% of hospitalized patients with SCD suffer from acute and chronic liver morbidities (24, 26). Individuals differ greatly in terms of the extent of liver involvement and the rate of progression of liver disease. As there are no definitive clinical characteristics or clinical laboratory tests for predicting or early diagnosis of SCD-associated hepatobiliary damage, people living with SCD must undergo regular monitoring. SCD has several genotypes, each with a unique presentation and clinical course (27). Hb SS, Hb SC, Hb Sβ⁺-thalassemia, and Hb Sβ⁰-thalassemia are the most common genotypes associated with SCD (28). Among them, HbSS is more frequently linked with liver dysfunction, whereas HbSC and HB-β thalassemia genotypes are less commonly associated with sickle-related liver problems (29, 30).

The commonly observed acute liver complications in SCD are acute sickle hepatic crisis, acute sickle intrahepatic cholestasis, acute hepatic sequestration, and acute gallstone disease. Acute sickle hepatic crisis occurs in ∼10% of patients with SCD and is characterized by transitory hepatic ischemia damage and vaso-occlusive crisis. Additional symptoms may include right upper quadrant pain, painful hepatomegaly, low-grade fever, and yellowing of the skin and eyes. Patients with SCD with acute hepatic crisis had a slight increase in liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which seldom surpass 1,000 IU/L, and bilirubin levels of less than 15 mg/dL (31).

The primary pathophysiology of acute sickle hepatic sequestration is hepatomegaly due to the sequestration of a large number of sickled erythrocytes in the hepatic vasculature and spleen, as well as acute anemia. The clinical symptoms are similar to acute sickle hepatic crisis, with considerable organ enlargement and a rapid drop in hemoglobin levels, which can lead to hypovolemic shock (32). Acute sickle intrahepatic cholestasis is a severe form of sickle hepatopathy with a high mortality rate. It is infrequent, primarily affecting people with homozygous HbSS genotypes (31, 33, 34). Transaminase elevations, hyperbilirubinemia, severe jaundice, renal failure, and coagulopathy are all present in this condition. The pathophysiology involves sickling of erythrocytes within sinusoids, which causes ischemia and hepatocyte injury, leading to hepatocyte ballooning and canalicular cholestasis (33–36).

Patients with SCD are at risk for chronic liver diseases. Recurrent episodes of hepatic ischemia with parenchymal tissue necrosis lead to hepatic fibrosis, chronic liver disease, and cirrhosis (31). Autopsy studies of people with SCD found that ∼16%–29% of them had cirrhosis. Patients with severe liver disease developed decompensated cirrhosis, which manifests as jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, or variceal hemorrhage (33, 37, 38). Sickle cholangiopathy is another consequence seen in people with SCD (39, 40) that causes biliary ischemia and biliary epithelium necrosis, resulting in fibrosis and strictures. Ascending cholangitis is a known cholangiopathy complication, as are bilomas and biliary abscesses in patients with SCD (38).

Other chronic liver problems that concern patients with SCD include viral hepatitis and iron overload from frequent blood transfusions (41–43). Hepatic iron overload has been identified as a significant concern in patients with SCD. Multiple blood transfusions cause iron deposition in the reticuloendothelial system and hepatocytes. Excess hepatic iron burden in patients with SCD might also be associated with ineffective erythropoiesis, which occurs less prominently than in patients with thalassemia (44, 45). Hemosiderosis can progress to cirrhosis in patients with SCD (46).

Nearly half of hospitalized patients with SCD experience hepatobiliary crisis (24), and there are no standard diagnostic or treatment approaches for liver problems associated with SCD. Based on autopsy and serum biochemical analysis, most studies found vaso-occlusion and hemolysis to be major pathophysiological processes in patients with SCD. The molecular mechanism of hepatobiliary damage in sickle liver is still unknown and requires further study to improve its treatment.

Over the past decade, mouse model studies have been especially useful in understanding the molecular events of SCD pathophysiology in great detail, which would otherwise be impossible using clinical research. Of note, SCD mouse research has aided in improved understanding of real-time vascular events leading to vaso-occlusive crisis (47), identification of various SCD pain receptors (48, 49), deciphering the involvement of coagulatory components in SCD disease manifestation (50), and hemolysis-driven organ injury (51). Several biomarkers and new mediators involved in vaso-occlusion, hemolysis, or communication between inflammatory cells and vascular endothelium were discovered in the SCD murine model using advanced imaging and molecular biology techniques such as real-time intravital imaging (52, 53), image flow cytometry, single-cell RNA sequencing, and other advanced imaging and molecular biology techniques, which was later validated by clinical research. Sickle mouse models also provide opportunities to intervene at different stages of development, which allows for the development of preventative therapies before the primary pathology’s effects lead to secondary complications. This review focuses on the molecular mechanism implicated in SCD-induced hepatobiliary damage using humanized knock-in mouse models of SCD.

Mouse Models of SCD

There are several humanized mouse models (both transgenic and knock-in models) with varying degrees of strength that can be used to investigate the pathophysiology and molecular mechanisms of SCD. Here, we briefly outline the mouse models widely used in SCD research. For a detailed description of SCD mouse models, the readers are referred to the following articles (54, 55).

HBSS; β-thalassemia mice.

The β-thalassemia mice are one of the oldest mouse models used in SCD research. This model was created by deleting the mouse β major gene while leaving the mouse β minor gene intact. Thus, their expression of mouse β globin genes is low. Using β-thalassemic mice as genetic background, transgenes expressing human HbS were introduced to generate several mouse models with varying levels of HbS (55). In these animals, the pathophysiology of the liver has not been studied in depth.

The New York sickle mice.

The New York (NY1DD) transgenic mice were one of the oldest transgenic SCD mouse model available, which has linked human α and β^S globin (αHβS) genes on a mouse β^major deleted background (56). These mice have a moderate SCD phenotype because they have 26% of sickle hemoglobin (HbS) levels. Previous studies have shown that these animals have inflammation and endothelial activation, which is increased by hypoxia/reoxygenation (H/R) (57, 58). Liver pathophysiology has not been investigated in depth in these animals and with the development of other lines with enhanced strength, NY1DD is no longer the model of choice for SCD research in mice.

S ± S^Antilles mice.

The S ± S^Antilles transgenic mice express human α-, β^S-, and β^S-Antilles globin transgenes (instead of mouse α and β genes) with a second mutation at β (23) position (valine to isoleucine) in addition to the β^S mutation on position β⁶ (56). These mice express ∼42% of human β^S and 36% of β^S-Antilles and show vascular pathology and inflammation that are further exacerbated by hypoxia (58, 59). Liver pathophysiology has not been analyzed in these mice.

HbSS-BERK mice.

When transgenic Berkley mice were introduced in 1997, it immediately gained tremendous popularity among SCD researchers (55). These mice are homozygous for knockout of both murine α and β globins and carry a single copy of the linked transgenes for human α and β^S globins. Compared with other weak models, HbSS-BERK mice express ∼99% human HbS, which makes it a suitable model for SCD murine research (55). Several studies in the past have shown presence of hepatic infarcts and liver injury at baseline in SCD BERK mice (50, 60, 61). However, the major drawbacks of the BERK mice are complicated genetic crosses to maintain the strain, which is both time-consuming as well as expensive and has a noisy background of five different strains. In addition, previous research has revealed the presence of a mild α-thalassemia phenotype in BERK mice, which does not exactly mimic the hematological model for most people with SCD who have all α-globin genes. Despite the drawbacks, SCD-BERK mice are routinely used and are a preferred model for many SCD researchers due to their ability to recapitulate SCD disease symptoms.

Townes knock-in mice.

Humanized Townes knock-in mice are an excellent model for studying SCD-induced hepatobiliary injury (25, 62, 63). The Townes SCD mouse model HbSS was created on a mixed genetic background by replacing mouse α and β globin genes with human α-globin genes and linked fragments of human β-sickle and fetal α-globin genes, respectively (62). Thus, the homozygous sickle Townes mouse carries mutations on the human hemoglobin α-gene [Hba^tm1(HBA)Tow, hα], the Hbb^{tm2(HBG1,HBB*)Tow} mutation, a 9.7-kb DNA fragment containing the human ^Aγ-globin gene, and human sickle hemoglobin beta (β^S) that substituted mouse major and minor β-globin genes (hα/hα::β^S/β^S). Heterozygous Townes mice carry the Hba^tm1(HBA)Tow (hα) mutation, one copy of human sickle hemoglobin beta (β^S), one copy of the Hbb^{tm3(HBG1,HBB)Tow} mutation, a DNA fragment containing human hemoglobin gamma (^Aγ) gene, and human wild-type hemoglobin beta (β^A) genes (hα/hα::β^A/β^S). Townes control animals carry two copies of the human α-globin gene and two copies of the Hbb^{tm3(HBG1,HBB)Tow} mutation (hα/hα:β^A/β^A).

HbSS Townes mice exhibit hepatic necrosis, vaso-occlusion, sterile inflammation, vascular congestion, cellular senescence, and increased iron overload within the liver (64, 65) (Fig. 2), mimicking the liver injury seen in patients with SCD and proving to be an excellent model for studying the liver dysfunctions associated with SCD (25). Using the Townes mouse model, this review gives an overview of current knowledge of the molecular mechanisms and potential treatment approaches for SCD-related liver dysfunction.

Figure 2.

Major liver pathophysiology associated with SCD. Schematic showing the chief liver pathophysiology associated with SCD. Increased hepatic inflammation (25), sinusoidal congestion (25, 53), iron overload (66), senescence (66), and exacerbated liver injury (25) are frequently associated with Townes SCD mice. SCD, sickle cell disease.

SCD Mouse Show Exacerbated Hepatic Inflammation

Townes SCD mice exhibit significant enrichment of inflammatory cells in the liver (Fig. 2), notably in periductular and parenchymal cells (25). Immunohistochemical and biochemical investigations have revealed a significant enrichment of hepatic resident macrophages (Kupffer cells) and neutrophils in the necrotic area of the liver of Townes SCD animal (25, 67), implying that both neutrophils and Kupffer cells are associated with SCD-induced liver injury (directly or indirectly). However, whether the increase in inflammatory cells is a cause or effect of liver injury remains to be elucidated. In addition, the levels of proinflammatory cytokines (TNFα, IL-1β, IL-2, and IL-6) are much higher in SCD mouse livers than those in control mice. This was in accordance with previous reports that showed increased inflammation in Townes SCD mouse (47, 68). Together, these observations hint at inflammation as important pathophysiology contributing to sickle-related liver dysfunction.

The role of inflammation in SCD-associated organ injury is not completely understood. Several literature reports suggest that the activation of inflammatory response in the Townes SCD mouse is associated with organ dysfunction (25, 69). However, recent studies show that the absence of tissue-resident inflammatory components is associated with more severe progression in the organ injury (66, 67). This demonstrates the complexity of activation of specific inflammatory components, which can generate either a damaging or protective response.

It is worth noting that the inflammation seen in SCD mice is classified as sterile inflammation. In contrast to inflammation, which is the body’s natural defense response to microorganisms and injury, sterile inflammation occurs in the absence of microorganisms and is generally associated with inflammatory signaling receptors releasing intracellular content from damaged or necrotic tissues. Mechanistically, sterile inflammation associated with SCD can cause tissue necrosis and the release of intracellular contents such as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) causing tissue injury (4). As such, it remains unclear how immune cells such as neutrophils, macrophages, and other innate immune cells that are recruited to the site of sterile inflammation site release DAMPs and PAMPs to promote inflammation and injury, which should be the focus of future studies in SCD mouse liver.

Sterile inflammation-induced recruitment of immune cells is also necessary for wound healing (70). The intercommunication between immune cells to ensure coordination of tissue healing through the clearance of dead cells and debris in SCD also remains unelucidated. Future studies using genetic knockouts or pharmacological suppression of immune components will be critical in understanding the involvement of inflammation in SCD liver damage.

NF-κB Pathway and SCD-Associated Hepatic Dysfunction

A significant increase in NF-κB and its downstream targets was observed in the livers of SCD mice (25). The NF-κB pathway plays a central role in liver inflammation and injury, including liver fibrosis (71). In the liver, cytokines and chemokines (such as TNFα and TLR of IL-1) are the main producers of NF-κB (71). The activation of NF-κB leads to the transcription of hundreds of genes, the majority of which are involved in the control of inflammation, immunological responses, and cell survival (71). Previous research has shown a specific role of NF-κB in specific hepatic cell types (71). It is critical to better understand the effect of NF-κB activation in distinct cell types of the SCD liver. It would also be important to tease out the role of NF-κB activation in SCD-associated liver apoptosis, inflammation, and wound healing.

Of interest, blocking NF-κB in a Townes SCD mouse model attenuated vaso-occlusion, partially ameliorated liver injury, and reduced both ALT and direct bilirubin levels (25). This suggests that NF-κB could be a crucial link between hepatic damage, fibrosis, and inflammation in SCD and could be a therapeutic target to ameliorate SCD-induced liver pathophysiology. Liver-specific knockouts of NF-κB pathway components can also be useful. However, the NF-κB node can be a double-edged sword (72), and inhibiting NF-κB may have not only therapeutic benefits but also a deleterious effect on hepatocyte viability, especially when NF-κB inhibition is severe in SCD. Finding appropriate targets that either have a mild effect on NF-κB activity or can be administered spatially and temporally to specific liver cell types will be critical to avoiding the injury associated with complete NF-κB inhibition.

Hepatic Iron Accumulation in Townes SCD Mouse Liver

Hepatic iron accumulation is a common phenotype seen in patients with SCD with repeated blood transfusions and is associated with increased mortality (73–76). Chronically transfused patients with SCD develop severe iron overload in liver, heart, spleen, and endocrine organs (34, 75–78). Free iron generates reactive oxygen species (ROS), which can promote cellular and organ damage (79). However, baseline changes in hepatic iron metabolism and homeostasis due to ongoing hemolysis and ineffective erythropoiesis are relatively less understood in SCD.

Townes SCD mice show hepatic iron accumulation in the central and mid-zonal regions of the liver at baseline (Fig. 2) (25). Interestingly, aging further exacerbates hepatic iron accumulation in Townes SCD mouse (67). Iron overload in the liver can eventually lead to fibrosis and cirrhosis progression (80). Thus, the chronic liver injury seen in Townes SCD mice could be predominantly due to hepatic iron overload. It would be interesting to evaluate the underlying reason for baseline iron enrichment in Townes mouse livers, as SCD-related anemia, inflammation, hemolysis, or hypoxia each can potentially cause hepatic iron accumulation. Moreover, any differences in hepatic iron content in male and female Townes SCD mouse should be analyzed in future studies as sex has also been shown to modulate iron homeostasis in mice (81, 82).

Iron levels in the liver are chiefly regulated by the hepcidin-ferroportin axis (83–85). Hepcidin levels are affected by inflammation (86), hypoxia (87), erythropoiesis (88), and bone morphogenetic protein (BMP) signaling (86, 89, 90). The absence of hepcidin results in hepatic iron accumulation, decreased iron in macrophages, and elevated serum iron that mimics human hereditary hemochromatosis. On the other extreme, excessive expression of hepcidin is associated with tissue iron overload, decreased iron absorption, and iron deficiency anemia. Earlier studies on SCD human samples have shown a low urinary hepcidin level in children (91). However, serum hepcidin level in adult patients with SCD was found to be highly variable, many of them showing a value of lower hepcidin than controls (92). Interestingly, studies with a significant history of transfusion and iron overload showed an increase in hepcidin level (44, 75, 77, 78, 93). These studies were limited by a narrow range of patient selection and importantly without any mechanistic insight. Future research concentrating on hepcidin regulation in SCD Townes mouse liver will be important for both mechanistic and therapeutic advances.

Similarly, mutations in ferroportin can also severely affect tissue iron homeostasis (83, 94, 95). Previous research has shown the presence of the ferroportin Q248H mutation in African populations with an allele frequency of 2.2%–13.4%, which is associated with a mild increase in serum ferritin and impaired iron homeostasis (96). Patients with SCD who carry this ferroportin Q248H variant have significantly higher levels of circulating interleukin-6 and C-reactive protein (97). Further research to determine the presence of this mutation in patients with SCD in different study cohorts and its effect on iron homeostasis and tissue iron overload would be useful.

Iron chelation therapy with deferoxamine has been found to improve iron excretion and eliminate extra tissue iron in patients with sickle cell disease, who are frequently transfused (79, 98). Regular chelation therapy lowers iron-related organ damage and mortality, according to long-term studies using deferoxamine in other hemoglobinopathies. It would be fascinating to investigate in more detail how deferoxamine affects molecular pathways of hepatic iron overload and liver damage in SCD mice over time. Therapy with hemopexin or synthetic ferroportin inhibitors also has the potential to reduce iron overload (65, 69, 99, 100). Heme-induced increased oxidative stress can be reduced by hemopexin (51, 101). Hemopexin was linked to the promotion of heme recovery and detoxification by the liver, primarily through the induction of HO-1 activity, and was associated with decreased heme-iron loading in the cardiovascular and kidney tissue, thereby decreasing oxidative stress in the endothelium with decreased induction of adhesion molecules (102). The effect of hemopexin or its mimetics on SCD-induced liver impairment will be fascinating to investigate. It will also be useful to study the effect of hemolysis-reducing drugs such as voxelotor (103) in SCD-induced hepatic iron accumulation and overall liver morbidities in Townes mice.

Liver Senescence Is Seen in SCD Mouse

Senescent cells are often observed in fibrotic and cirrhotic livers (104). The p53 transcription factor plays a critical role in cellular senescence through p21 induction (105–107). Moreover, senescent cells upregulate the expression of inflammatory cytokines and are prone to apoptosis through NK cell-mediated killing, which limits fibrosis progression (108). Remarkably, along with an increase in inflammation and cytokine production, Townes SCD mouse also exhibits a significant increase in liver senescence at baseline (Fig. 2) (25) as seen by increased expression of the senescent markers p53, p21, and p16 in the liver (64).

Enhanced expression of senescent cell markers (P53, P21, and P16) is associated with the polarization of liver macrophages to the M1 state through their senescence-associated secretory phenotype (SASP) (109). Consistently, significant enrichment of M1-specific macrophages was found in Townes SCD mouse livers. Intriguingly, increased hepatic iron accumulation was also observed in senescent cells. Iron can induce liver senescence through the STAT3-p53 axis, limiting liver fibrosis (110). Thus, it will be necessary to understand the role and regulation of senescence in liver fibrosis seen in SCD. Previously, it was observed that the liver of Townes SCD mouse had substantial apoptosis. Therefore, understanding and distinguishing between apoptosis and senescent cells in SCD liver are critical. It is also crucial to compare the presence of senescent cells in both the sinusoidal and extra sinusoidal compartments of the liver (25).

In sum, the role of senescence in SCD liver fibrosis is still unresolved and requires further experiments using cell-specific genetic modifications as well as experimental models of SCD-associated liver fibrosis.

Other Miscellaneous Phenotypes Seen in SCD Mouse Liver

Biliary injury in SCD mouse liver.

Sickle cell intrahepatic cholestasis (SCIC) is one of the causes of liver failure in patients with SCD (111, 112). SCD mouse also exhibits increased ductular response accompanied by increased levels of total bile acid in the liver at baseline (Fig. 3) (64). Mechanistically, it was shown that NF-κB-driven chronic inflammation leads to the downregulation of FXR and its targets, which in turn might lead to delayed bile transport (Fig. 4). However, overexpressing FXR did not cause a very significant amelioration of liver injury in SCD mice (unpublished data), suggesting alternate routes of bile secretion mechanisms in SCD mice that need to be evaluated in future research. The bile transport defect seen in SCD mice could be predominantly inflammation-driven, and treating inflammation for a prolonged period could be useful to ameliorate this defect. In addition, the effect of hypoxia on bile transport defects in SCD should also be assessed.

Figure. 3.

Biliary pathophysiology in SCD liver. Schematic showing the cascade of events leading of biliary pathophysiology in SCD mouse liver, including delayed bile flow and toxic bile (25). SCD, sickle cell disease.

Figure 4.

Major signaling pathways associated with SCD-associated hepatic dysfunction. Schematic showing the network of known signaling pathways associated with SCD-associated liver dysfunction. SCD, sickle cell disease.

Vascular congestion is seen in SCD mice.

Townes SCD mouse exhibit sinusoidal ischemia at baseline (observed by multiphoton excitation enabled in vivo real-time fluorescence microscopy of the intact liver in live mice as well as by gross liver morphology of Townes SCD mouse) (25). Interestingly, no other organs have been reported to show vaso-occlusion at baseline (113). This suggests that the liver might be one of the most susceptible organs to vaso-occlusion-induced ischemic injury. This might also hint at oxygen depletion as a major source of liver injury seen in Townes SCD mouse.

Reversibility of SCD-associated liver injury: lessons from mouse model.

Previous research by others and us indicates that addressing one or more of the major underlying pathologies, such as inflammation, iron overload, bile transport deficiency, hypoxia, vaso-occlusion, or hemolysis, can aid in the reversal of SCD-associated hepatobiliary injury. The mouse model can be especially valuable in evaluating the short- and long-term effects of current and potential therapeutic choices in the liver and other organs and should be studied extensively. One recent example of using the SCD mouse model to study the long-term effects on the liver was performed by deleting the P-selectin gene in SCD mice (25, 114).

P-selectin, a cellular adhesion protein expressed on the endothelium and activated platelets (115–117), is a promising therapeutic target for SCD-induced vaso-occlusive crisis (114, 118). P-selectin regulates leukocyte capture, rolling, and recruitment on active vascular endothelium, as well as platelet-leukocyte interactions (115, 117, 119, 120). As of November 15, 2019, the US Food and Drug Administration approved crizanlizumab, a humanized monoclonal antibody against P-selectin, for reducing the frequency of vaso-occlusive crises in patients with SCD (121).

Recently, it was found that despite improvements in liver sinusoidal vaso-occlusion, ischemia, and transaminases, the absence of P-selectin does not prevent hepatobiliary injury in Townes SCD mice (67, 114). The exacerbated liver injury and senescence seen in P-selectin-deficient SCD mice were attributed to reduced iron removal in the liver and poor leukocyte migration into the hepatic tissue (67). Hemolysis did not improve based on liver heme oxygenase-1 levels, hemoglobin, or reticulocyte numbers in P-selectin-deficient SCD mice (25). This study reveals a potential risk of complete loss of P-selectin on delicate physiological balances. This risk might be alleviated by the incomplete inhibition of P-selectin afforded by current therapeutic interventions.

Another important pathophysiology of SCD mouse liver was exacerbated inflammation and activation of NF-κB signaling (Fig. 4) (67). However, blocking NF-κB using the general antioxidant N-acetylcysteine only partially ameliorated SCD-associated liver injury, suggesting that additional factors also contribute to hepatobiliary injury in SCD (25). Use of antioxidants to alleviate SCD-induced oxidative stress may also be a viable option for ameliorating liver injury and should be investigated in the Townes mouse model. Similarly, the effectiveness of anti-inflammatory drugs in preventing SCD-associated liver problems should be examined in this model.

Among other interesting phenotypes, the loss of canalicular bile transporters in Townes mice liver may have potential therapeutic implications (25). However, the cause-and-effect relationship between these pathological events is still unknown and should be investigated further in future research. Mouse study also showed an increase in taurine (mice)- and glycine (human)-conjugated secondary bile acids in SCD (25), which has been previously shown to be associated with prolonged exposure to antibiotics resulting in loss of gut microbiota (122). Interestingly, an aberrant gut microbiome was recently shown to promote inflammation and systemic ischemia in SCD mice (123). Thus, future investigations should explore the role of the microbiome and altered composition of bile acids in promoting hepatobiliary injury in SCD.

CONCLUSIONS

Given the global burden of SCD and the lack of preclinical alternatives, mouse models are critical for further elucidating the pathophysiology of SCD development and SCD-associated chronic organ injury, as well as defining viable therapies and possible biomarkers. Using a knock-in humanized Townes mouse model of SCD, this review revealed new insights into the cellular and molecular underpinnings of SCD-associated hepatopathophysiology. The Townes humanized SCD mouse model, like patients with SCD, displays a wide range of liver pathologies, from inflammation to cholestasis to fibrosis and hepatic iron accumulation (64, 67, 114) and thus a useful model to study the pathophysiology and molecular mechanism (Fig. 3) of SCD-induced hepatic dysfunctions. Mechanistic studies in Townes mice can aid in the identification and development of novel biomarkers for liver dysfunction in patients with SCD. In addition, this model can be used to examine the effect of currently approved Food and Drug Administration drugs on liver toxicity in SCD. In summary, the humanized Townes knock-in model of SCD is an excellent tool for studying the molecular mechanisms and for identifying new biomarkers and therapeutic agents for SCD. Future and ongoing fundamental research using this model will validate the efficacy, specificity, and safety of novel therapeutic options for hepatopathophysiology associated with SCD.

GRANTS

This work was supported by a Scholar Award from the American Society of Hematology and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK125617 to T.P-S.

DISCLOSURES

G.J.K. works at CSL Behring. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

T.P-S. conceived and designed research; T.P-S. prepared figures; T.P-S. drafted manuscript; T.P-S., G.J.K., and E.M.N. edited and revised manuscript; T.P-S., G.J.K., and E.M.N. approved final version of manuscript.