Evidence of Immunological Abnormalities in Sitosterolemia

Review Article

Austin Med Sci. 2021; 6(2): 1053.

Evidence of Immunological Abnormalities in Sitosterolemia

La Rosa A1,2*, Ivaldi F1, Penco F3, Bocca P3, Volpi S1,3 and Di Rocco M2

¹Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics and Maternal and Child Sciences - DiNOGMI, University of Genova, Genoa, Italy

²Department of Pediatric Sciences, Rare Diseases Unit, IRCCS Giannina Gaslini, Genoa, Italy

³UOSD Center for Autoinflammatory Diseases and Immunodeficiencies, IRCCS Istituto Giannina Gaslini, Genoa, Italy

*Corresponding author: Alessandro La Rosa, Department of Neuroscience, Rehabilitation, Ophthalmology, Genetics and Maternal and Child Sciences - DiNOGMI University of Genoa Largo Paolo Daneo, 16132 Genova (GE), Italy

Received: September 07, 2021; Accepted: October 07, 2021; Published: October 14, 2021


Objectives: Mutations involving the ABCG8 gene cause sitosterolemia (STSL), a rare metabolic disease due to an abnormal plasmatic plant sterols level. STSL hematological findings include stomatocytic hemolysis and macrothrombocytopenia. So far, few cases of leukopenia have been reported and there are no studies that consider inflammatory activity. The purpose of our study is to evaluate immunological abnormalities in STSL and the effect of dietary and pharmacological treatment.

Study Design: In a sitosterolemic patient presenting leukopenia and increased inflammatory indexes were evaluated in vitro interleukin 1B secretion from monocytes and type 1 interferon (IFN) signature. Besides, Stimulation Indexes (SI) of patient’s and two Healthy Controls’ (HCs) T-helper lymphocytes in response to proliferative stimuli were assessed, when incubated in enriched medium alternately with patient plasma or with AB Rh+ group control plasma. SI (compared to the same HCs) and IFN signature have been evaluated again three (T3) and six (T6) months apart, after patient received therapy.

Results: Following treatment with Ezetimibe (EZE) and adequate diet, lower plasmatic plant sterols levels and progressive improvement of hematological and immunological abnormalities were observed. These data were confirmed by the negativization of IFN signature at T3 and by the progressive normalization at T3 and T6 of SI in patient’s and HCs’ T-helper lymphocytes incubated in patient’s plasma-enriched medium.

Conclusion: High plasmatic phytosterols levels due to STSL seems to affect white blood cells proliferation and trigger pro-inflammatory process, justifying leukopenia and increased phlogosis indexes observed in our patient. These hypotheses are supported by normalization of clinical and laboratory data during EZE therapy.

Take-home Message: Immunological abnormalities should be considered among the hematological manifestations of sitosterolemia, moreover high levels of plasma phytosterols could be a pro-inflammatory trigger. Treatment with ezetimibe seems to reverse these manifestations.

Keywords: Sitosterolemia; Inflammation; Plant sterols; Immunological abnormalities; Type I interferon signature; ABCG8


Hb: Hemoglobin; PLT: Platelet Count; MPV: Mean Platelet Volume; WBC: White Blood Cells; HDL: High Density Lipoprotein; LDL: Low Density Lipoprotein; CRP: C-Reactive Protein; ESR: Erythrocyte Sedimentation Rate


The mechanism of intestinal mucosa transporters and regulating systems of plant sterols’ absorption have been recently described. Physiologically, plasma levels of phytosterols are controlled by a balance between the absorption and efflux systems represented by the transporters sterolin-1 and -2 [1]. These efflux pumps, expressed on enterocytes and biliary canalicular membranes, contain ATP Binding Casette (ABC) proteins encoded by ABCG5 and ABCG8 [2]. Homozygous or compound heterozygous mutations of these genes lead to a rare disease named Sitosterolemia (STSL), characterized by an excessive accumulation of phytosterols, especially β-sitosterol. Clinically it presents huge phenotypic heterogeneity from completely asymptomatic forms to advanced atherosclerosis and premature cardiac death [3,4], Patients are also known to present immunological abnormalities and signs of systemic inflammation [5]. Alteration of lipid metabolism might influence inflammatory pathways, the most relevant example being deficit of sterol synthesis pathway due to mevalonate kinase deficiency, an autosomal recessive inherited metabolic disease clinically characterized by recurrent fever and elevation of inflammatory markers due to dysregulation of the inflammatory cytokine secretion interleukin 1 beta (IL-1 β) in monocytes [6]. Of note, macrophage activation via toll-like receptors (TLR) 4 agonist is modulated by β –sitosterol [7].

Characteristic hematologic manifestations of STSL include stomatocytic anemia, macrothrombocytopenia, splenomegaly and abnormal bleeding [8]. So far, only anecdotal cases of leukopenia have been reported [9] and there are no studies that consider inflammatory activity, which is often described in patients with STSL. Treatment with Ezetimibe (EZE) allows reduction of plasma levels of phytosterols and so the normalization of platelets count, Mean Platelet Volume (MPV) and Hemoglobin (Hb) levels [10], improving prognosis and long-term outcome [11].

The aim of our study is to focus on immunological abnormalities in sitosterolemia and the reverse after dietary and pharmacological treatment.

Study Design

A 17-year-old patient presenting a history of mild normocytic anemia and thrombocytopenia, hypergammaglobulinemia, hyperferritinemia, increased inflammatory indexs, arthralgias, partially responsive to analgesic therapy, and xanthomas on the extensor surfaces of joints was evaluated for STSL. Diagnosis was genetically confirmed by the presence of homozygous variant c.490 C> T p. (Arg164) of ABCG8 gene causative for STSL [12]. After obtaining informed consent, the patient was enrolled in the study. At diagnosis (T0) Hb, Platelet count (PLT), MPV, White Blood Cells (WBC), lipid profile and analysis of plasmatic phytosterols were evaluated.

To investigate the inflammatory state, C Reactive Protein (CRP), Erythrocyte Sedimentation Rate (ESR), peripheral blood type I interferon (IFN) signature and in vitro IL-1 β secretion from monocytes were evaluated. IFN signature [13] and in vitro IL-1 β secretion [14] have been performed as described. Briefly, for IFN signature, RNA was extracted from peripheral blood collected in PAXgene tubes using the PAXgene Blood RNA kit (Qiagen, Hilden, Germany). cDNA was retrotranscribed using SuperScript® VILO™ cDNA synthesis kit (Invitrogen, Carlsbad, California, USA). Selected IFN-stimulated gene (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) expression was quantified by real-time PCR using gene-specific primers and probes (Roche) with the ddCt method relatively to a healthy donor calibrator using HPRT and G6PD as reference genes.

Peripheral Blood Mononuclear Cells (PBMCs) were harvested by density gradient from blood collected in heparin tubes. To assess IL-1 secretion, monocytes were isolated by cell adherence after one-hour incubation in 24 well plates and stimulated with Lipopolysaccharide (LPS) 100ng/ml; supernatants were collected before and after three, six and eighteen hours of stimulation; IL-1 β was measured by ELISA (R&D). To study lymphocyte proliferation, PBMCs were stained with Carboxyfluorescein Succinimidyl Ester (CFSE) [15]. 400.000 PBMCs were dispensed on a 96 well plates. Patient’s PBMCs were incubated in complete RPMI 1640 medium enriched alternately with either 10% of the patient’s plasma or 10% of group AB Rh+ control plasma and stimulated with phytohaemagglutinin (PHA 1μg/mL, Sigma, St. Louis, MO) or anti-human CD3 and anti-human CD28 soluble (5μg/ mL each, BD, USA). This procedure was performed simultaneously on two Healthy Controls’ (HCs) PBMCs, selected by age and blood group homologous to the patient.

After four days of incubation, flow cytometry analysis was used to assess patient and HCs T-helper lymphocyte proliferation in response the different plasmas, using surface markers aCD3 (BV 420, BD, USA), aCD4 (APC, BD, USA) aCD8 (PE-Cy7, BD, USA) [16] The Stimulation Index (SI) was estimated as ratio of proliferated cells to unproliferated as a measure of CFSE response [15] This procedure was performed to assess the interference of high levels of circulating phytosterols with cell proliferation.

Blood tests, plasma plant sterols levels, SI (compared to the same HCs) and IFN signature have been valuated again three months (T3) apart, after patient received dietary and EZE therapy. SI and IFN signature were repeated one more at six months (T6) of treatment.


We have analyzed hematological response to the dietetic and pharmacological treatment in a patient with a new diagnosis of STSL. At baseline, in vitro secretion of IL-1 β by monocytes following TLR4 stimulation was moderately increased compared to HCs (Figure 1). Table 1 reports the hematological findings at baseline and their variations at T3 and T6 after starting therapy with EZE. As expected, we assessed normalization of Hb and LDL, with progressive increase in WBCs count and reduction in plasmatic phytosterols about of 26% and inflammatory indexes (CRP and ESR).