INTRODUCTION:

Statins are a chemically related group used as lipid-lowering drugs. They reduce hepatic lipid biosynthesis by inhibition of hydroxy-methyl-glutaryl Coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol biosynthesis¹. Studies confirmed that statins have additional pleiotropic, cholesterol-independent, effects mediated by inhibition of isoprenoid synthesis with subsequent inhibition of downstream signaling molecules like Rho, Rac, and Ras². Results have also shown that chronic statin treatment, especially with the lipophilic chemistry, may adversely affect ubiquinone concentration, an important electron carrier in the mitochondrial respiratory chain³.

Such extra-hepatic effects impose the preferences of one statin over another⁴. Indeed, one of the most important pleiotropic effects of statins includes its anti-inflammatory action⁵, which is mainly linked to lymphocyte functionality, proliferation, and maturation⁶. Reduction of inflammation and modulation of immunity has been confirmed via mouse inflammation model and clinical trial of organ transplant rejection or multiple sclerosis⁷. Statin inhibits proliferation and cytotoxicity of an aggressive natural killer cell leukemia⁸, Effects of simvastatin on the function of splenic CD⁴⁺ and CD⁸⁺ T cells in sepsis mice⁹.

At a molecular level, the specific immunomodulatory target of statins is not clearly defined¹⁰. However, statin interferes with protein isoprenylation resulting in modulation of post-receptor translation pathways with resultant mislocalization of inactive Rac intermediate in the cytosol¹¹ with subsequent inhibition of NADPH oxidase enzyme and NADPH-derived free radicals (Figure 1)¹². Additionally, statins also interfere with cell membrane lipid raft resulting in loss of cellular integrity. However, Blank et al, have shown that atorvastatin inhibition of lymphocyte proliferation started from (0.2µM) to complete inhibition with (10µM) and the inhibition was not rescued with mevalonate incubation ¹³. Lymphocytes are a type of white blood cells that play a fundamental role in the immune response. The latter such an additional target at a molecular level in addition to their primary effects on LDL-cholesterol level¹⁴. For example, it may interfere with cell migration and localization through disrupting lymphocyte-endothelial communication reducing homing to the target site in tissues resulting in inhibition of lymphocyte extravasation¹⁵.

Figure 1. Statin inhibition of the mevalonate pathway. Statins inhibit isoprenoid intermediates and Rac activation.

Lymphocyte functionality is affected by targeting antigen-presenting cells through inhibiting the interaction of the T-cell receptor with major histocompatibility complex protein¹³. It has been confirmed that statin reduces INF release affecting the assembly of major histocompatibility complex protein on the surface of antigen-presenting cells. Other studies have shown that statin inhibits the surface costimulatory protein on antigen-presenting cells which makes the recognition by T cells incomplete; these surface molecules include integrins (LFA-1 and ICAM-1) ¹⁵ (Figure 2). Results have also shown that ubiquinone level was reduced in lymphocytes with three months of statin treatment, thus lymphocyte mitochondria could be another target for statin anti-inflammatory action of statin treatment.

Figure 2. Effects of statins on T-cell activation and differentiation. By inhibition of isoprenoid intermediate synthesis, statins suppressors T-cells activation, they also inhibit antigen uptake, processing, and presentation, as well as the expression of MHCII.

It is well known that chronic statin treatment has strong anti-inflammatory effects by the inhibition of isoprenoid intermediates like the Ras-GTPase family which are the cornerstone in activating cell surface receptors and downstream signaling mechanism in the inflammatory cells like lymphocytes, macrophages, and endothelial cells. However, the acute effect of the lipophilic statins in comparison with the hydrophilic statins is still not clearly defined, Moreover, studies have also shown that some pleiotropic effects of statins are related to their structural diversity and their physicochemical properties ¹⁵, for example, T cells activation were inhibited by the lipophilic lovastatin and simvastatin while no effects were reported by the treatment with the hydrophilic pravastatin. Therefore, the current study aimed to examine the effect of short-term treatment with lipophilic atorvastatin in comparison with the relatively water-soluble rosuvastatin on lymphocyte count in hyperlipidemic patients.

MATERIALS AND METHODS:

Patients and Methods:

Patients were recruited from a private clinic and randomly categorized into atorvastatin 20mg per day (15 patients) or rosuvastatin 20mg per day (15 patients) for 6 weeks period. The study design was a randomized, single-blind study. All patients included in the study were characterized with hypercholesterolemia by laboratory test of lipid profile. Patients have given atorvastatin 20mg or rosuvastatin 20mg once daily during a 6- weeks treatment period. C-reactive protein (CRP) tests were done for all patients to exclude any sort of inflammation giving false-positive results and only those patients with normal CRP were enrolled in the study (Table 1). Patients were nearly matched regarding their demographic factors, such as age, sex, and BMI (Table 1).

Table 1: Demographic characteristics of studied groups

Parameters	Treated groups
Parameters	ATG (n=15)	RTG (n=15)
Gender (male: female)	6:9	7:8
Age (year)	43-55	48-57
BMI (kg/m²)	27.5	26
Smoking: not smoking	0:15	0:15
CRP (mg/L)	9-11
ATG: Atorvastatin-treated group RTG: Rosuvastatin-treated group CRP: C-Reactive Protein	BMI: body mass index kg: kilogram m²: square meter

Exclusion Criteria:

Patients with apparent hepatic disease, any sort of renal disease, diabetes mellitus, cardiovascular disease, cancer, inflammatory diseases, or on any medications which are well-known to be associated with leukopenia, or bone marrow disorders. Were excluded from the study. No patient had taken any lipid-lowering agent during the last 2-months before our study.

CRP measurement:

CRP was measured by ELISA techniques. The technique simply involves using sandwiched antibodies to determine the concentration of CRP. The standard and serum samples were loaded to overnight precoated 96-well plates with capture antibodies and incubated for 2h with gentle shaking. The third step was the addition of a diluted biotin-labeled antibody and incubated with gentle shaking for 1 h. Enzymatic reactions were started by the addition of streptavidin. After 2h incubation period, the enzymatic reaction was terminated by the addition of detection antibodies. Subsequently, the diluted streptavidin-HRP conjugate was added and incubated for 45 min. Finally, a substrate was added and incubated for 30 min with gentle shaking producing blue color which turns to yellow upon addition of stop solution, and the optical density determined using a plate reader at 450nm. It's worthy to mention that each one of these individual steps was followed by washing the plate 4 times by washing buffer and the whole assay procedure were taken place at room temperature.

Differential WBC count:

A complete blood picture was conducted on the blood samples collected on K2EDTA-treated tubes (Terumo Europe NV, Belgium); the blood sample analysis was conducted by an ADVIA 2120 fully-automated analyzer (Siemens Health-care Diagnostics, USA). The local reference percentage of samples were mentioned in table 2 (see below). Data expressed as the mean ± standard deviation. Groups were compared results by paired t-test. *P<0.0001 was used to describe a statistically significant difference. GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA) was used to obtain the results.

Table 2. Normal WBCs reference range.

Type of WBC	Percentage
Neutrophil	40-760
Lymphocyte	20-40
Eosinophil	1-4
Monocyte	2-8
Basophils	0.5-1

RESULTS:

Atorvastatin significantly changed decreased LDL in comparison with the pretreatment plasma LDL levels (***P<0.001) (Figure 3).

Figure 3. Effects of 6-weeks of treatment with A-atorvastatin B-Rosuvastatin on HbA1c. Data are expressed as the mean ± standard error. **P<0.01, ***P<0.001.

The present study confirmed that short-term use of atorvastatin was associated with a significant reduction of lymphocyte percentage (lymphocyte percentage before 40.2±7.06 versus after 32.3±6.02) (p<0.0001), by contrast, rosuvastatin had no significant effect on lymphocyte count (lymphocyte percentage before 32.1±4.7 versus after 30.3±6.1), however, there was no significant change with neutrophils count (Figure 4). This reduction was noticed in each sample of the 15 atorvastatin-treated subjects included in the study (Figure 6). In addition to that other differential cells (Eosinophil, Monocytes, and Basophils) were shown no change in both groups (Figure 5).

Figure 4. Short-term use of atorvastatin reduced lymphocyte count. Differential cell count, before and after atorvastatin and rosuvastatin used for a month has been associated with a significant reduction in lymphocyte count and no change in neutrophil count in the atorvastatin group compared to the rosuvastatin group. data expressed as mean±SD (n=15), ***p<0.0001, NS=non-significant.

Figure 5. Short-term use of atorvastatin and rosuvastatin does not affect eosinophil, monocyte, and basophil. Differential cell count, before and after atorvastatin and rosuvastatin used for a month has been associated with a non-significant change in either eosinophil, monocyte, or basophil count in both atorvastatin and rosuvastatin group. data expressed as mean±SD (n=15), NS=non-significant.

Figure 6. Individual patient lymphocyte count in the atorvastatin group. Lymphocyte count before and after atorvastatin used for a month has been associated with a reduction in lymphocyte count in each patient.

DISCUSSION:

The present study showed that short-term use of atorvastatin was associated with a significant reduction of lymphocyte percentage while there was no significant change with neutrophils count. By contrast, rosuvastatin had no significant effect on lymphocyte count (Figure 4), such result suggested that statin affect lymphocyte specifically comparing to other white blood cells. Results suggested that the effects are either at cellular levels or even at molecular levels. Some of these reports confirmed that the secretome profile was changed after statins or the immunological response was modulated in other studies¹⁶.

There is no clear evidence to explain this selectivity between the white blood cells. One thing which should be considered is the lineage diversification between these cells during hematopoietic development inside bone marrow. Interestingly, lymphocytes originated from lymphoid lineage while the rest were originating from myeloid lineage¹⁷. This variability linked with the variation in lineage commitment influenced by various factors, and some of these might be affected by statins. It's worthy to mention that another lymphoid-lineage committed natural killer cells greatly affected by statins confirming such hypothesis¹⁸. Analysis of result obtained from an experimental study of statins added to co-culture system involved IL-2- exposed natural killer cells and cancer cells (K562 cells) showed modulation of cytotoxic activity of natural killer cells.

Interestingly simvastatin and fluvastatin (lipophilic) inhibit the cytotoxic effect while pravastatin (hydrophilic) did not¹⁸. Moreover, the inhibitory activity of simvastatin and fluvastatin was partially restored upon the addition of the intermediary product of cholesterol synthesis, mevalonate¹⁸. Instant in vitro activation, with subsequent proliferation, of freshly-isolated lymphocytes, has been inhibited by atorvastatin.

An in vitro T cell line model (Jurkat cells) was co-cultured with activated peripheral blood mononuclear cells (PBMC) in the presence and absence of atorvastatin¹³. The result has shown that Jurkat cell proliferation was inhibited in presence of atorvastatin compared to the control sample. In addition to that, the cell secretory property has been modulated in presence of statin; IL-2 main secretome component of Jurkat cells and many other immune cells is inhibited by statin compared to the control sample.

Third-party-stimulated or IFN-r stimulated T cells were not affected by atorvastatin reducing the possibility for statins to find an application in organ transplantation¹³.

Another proposed mechanism of statin reduction of lymphocyte concentration is the inhibition of clonal expansion following activation. The mechanism of action is based on statin reduction of proliferation of T lymphocytes and their selective direction toward Th2 rather than Th1 proliferation¹⁹.

The lymphocytes were characterized by higher mitochondrial density compared to other immune cell types²⁰. Statins disrupt cellular function at their cytoplasmic organelles level; mitochondria in particular ²¹. Previous studies have confirmed mitochondrial depolarization by the lipophilic statins in different cell types like skeletal muscle, hepatic cells, and pancreatic β-cells²², Since, the present results confirmed reduced lymphocyte level after short-use of the lipophilic atorvastatin; thus the mechanism could be linked to mitochondrial disruption. However, this effect should be considered at an early stage of the hematopoietic tree in the bone marrow which might confirm that statin could have a role in directing lineage preference during cellular commitment.

Despite that each sample show lymphocyte count reduction, still there is interindividual variation. We noticed that 8 out of 15 patients show a very good reduction in lymphocyte count and 7 of them show a slight reduction. This variation could be part of normal interindividual differences regarding their demographic parameters. Unfortunately, there are limited published data in this regard for comparisons. However, the variation could be also linked to heterogeneity of the population of lymphocytes during activation, proliferation, or maturation. T lymphocyte clonal expansion following activation takes place variably in various subjects and statin effects reached correspondingly. The interference of different statin members with the clonal expansion is confirmed through a comparison of rosuvastatin and atorvastatin²³^,²⁴.

The inconsistent effect of statin on immune cells might be explained in terms of the ratio of dominant cells (neutrophils and lymphocytes) while others (eosinophils, monocytes, and basophils) are presented only in a limited count (table 2)²⁵. Moreover, the lymphocyte is characterized by clonal expansion; an effect that is absent in other cells which are derived from myeloid lineage²⁴. Nevertheless, in vitro incubation of myeloid cells with statins is associated with the modulation of their secretory profile¹³^,¹⁵^,40.

CONCLUSION:

Statins have systemic anti-inflammatory effects reducing lymphocyte percentage in the peripheral circulation. The result shows that this effect was specifically associated with atorvastatin (lipophilic) rather than rosuvastatin (hydrophilic) at equivocal dose despite confirmed effectiveness of rosuvastatin at lower doses compared to atorvastatin.

LIMITATION:

The limitation of our study includes small sample size and ignorance of other statins (simvastatin, pravastatin, and pitavastatin). Further studies should be conducted at the cellular and molecular levels to identify the site of interference which might be at the far deepest level reaching bone marrow and this will revolutionize the medicine at bone marrow transfer approaches finding a new application in controlling cellular proliferation in leukemic subjects.

ACKNOWLEDGEMENT:

The authors are grateful to College of Pharmacy and College of Medicine in University of Mosul and Ninevah University for their help and support.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Bonetti PO. Lerman LO. Napoli C. Lerman A. Statin effects beyond lipid lowering—are they clinically relevant?. European Heart Journal. 2003; 24(3): 225-48. doi.org.10.1016/S0195-668X(02)00419-0

2. Cordle A. Koenigsknecht-Talboo J. Wilkinson B. Limpert A. Landreth G. Mechanisms of statin-mediated inhibition of small G-protein function. Journal of Biological Chemistry. 2005; 280(40): 34202-9. doi.org.10.1074/jbc.M505268200

3. Qu H. Meng YY. Chai H. Liang F. Zhang JY. Gao ZY. Shi DZ. The effect of statin treatment on circulating coenzyme Q10 concentrations: an updated meta-analysis of randomized controlled trials. European Journal of Medical Research. 2018; 23(1): 1-0. doi.org.10.1186/s40001-018-0353-6

4. Sokalska A. Hawkins AB. Yamaguchi T. Duleba AJ. Lipophilic statins inhibit growth and reduce invasiveness of human endometrial stromal cells. Journal of Assisted Reproduction and Genetics. 2019; 36(3): 535-41. doi.org.10.1007/s10815-018-1352-9

5. Koushki K. Shahbaz SK. Mashayekhi K. Sadeghi M. Zayeri ZD. Taba MY. Banach M. Al-Rasadi K. Johnston TP. Sahebkar A. Anti-inflammatory action of statins in cardiovascular disease: the role of inflammasome and toll-like receptor pathways. Clinical Reviews in Allergy and Immunology. 2021; 60(2):175-99. doi.org.10.1007/s12016-020-08791-9

6. Sorathia N. Al-Rubaye H. Zal B. The effect of statins on the functionality of CD4+ CD25+ FOXP3+ regulatory T-cells in acute coronary syndrome: a systematic review and meta-analysis of randomised controlled trials in Asian populations. European Cardiology Review. 2019; 14(2):123. doi.org.10.15420/ecr.2019.9.2

7. Qin L. Xie X. Fang P. Lin J. Prophylactic simvastatin treatment modulates the immune response and increases survival of mice following induction of lethal sepsis. Journal of International Medical Research. 2019; 47(8):3850-9. doi.org.10.1177/0300060519858508.

8. Henslee AB. Steele TA. Combination statin and chemotherapy inhibits proliferation and cytotoxicity of an aggressive natural killer cell leukemia. Biomarker Research. 2018; 6(1):1-1. doi.org.10.1186/s40364-018-0140-0

9. Kong B. Wang X. Yang W. Zhao X. Zhang R. Wang Y. Effects of simvastatin on the function of splenic CD4+ and CD8+ T cells in sepsis mice. Immunologic Research. 2018; 66(3):355-66. doi.org.10.1007/s12026-018-8994-7

10. Akula MK. Ibrahim MX. Ivarsson EG. Khan OM. Kumar IT. Erlandsson M. Karlsson C. Xu X. Brisslert M. Brakebusch C. Wang D. Protein prenylation restrains innate immunity by inhibiting Rac1 effector interactions. Nature Communications. 2019; 10(1):1-3. doi.org.10.1038/s41467-019-11606-x

11. Adam O. Laufs U. Rac1-mediated effects of HMG-CoA reductase inhibitors (statins) in cardiovascular disease. Antioxidants and Redox Signaling. 2014; 20(8):1238-50. doi.org.10.1089/ars.2013.5526

12. Dos Anjos PM. Volpe CM. Miranda TC. Nogueira-Machado JA. Atorvastatin Inhibited ROS Generation and Increased IL-1β And IL-6 Release by Mononuclear Cells from Diabetic Patients. Endocrine, Metabolic and Immune Disorders-Drug Targets (Formerly Current Drug Targets-Immune, Endocrine and Metabolic Disorders). 2019; 19(8):1207-15. doi.org.10.2174/1871530319666190617160349

13. Blank N. Schiller M. Krienke S. Busse F. Schätz B. Ho AD. Kalden JR. Lorenz HM. Atorvastatin inhibits T cell activation through 3-hydroxy-3-methylglutaryl coenzyme A reductase without decreasing cholesterol synthesis. The Journal of Immunology. 2007; 179(6):3613-21. doi.org.10.4049/jimmunol.179.6.3613

14. Krysiak R. Okopien B. Effect of simvastatin on hemostasis in patients with isolated hypertriglyceridemia. Pharmacology. 2013; 92(3-4):187-90. doi.org.10.1159/000341909

15. Ghittoni R. Enea Lazzerini P. Laghi Pasini F. Baldari CT. T lymphocytes as targets of statins: molecular mechanisms and therapeutic perspectives. Inflammation and Allergy-Drug Targets. 2007; 6(1):3-16. doi.org.10.2174/187152807780077291

16. Mach F. Statins as immunomodulatory agents. Circulation. 2004; 109(21):II-15. doi.org.10.1161/01.CIR.0000129502.10459.fe

17. Prinyakupt J. Pluempitiwiriyawej C. Segmentation of white blood cells and comparison of cell morphology by linear and naïve Bayes classifiers. Biomedical Engineering Online. 2015; 14(1):1-9. doi.org.10.1186/s12938-015-0037-1

18. Tanaka T. Porter CM. Horvath-Arcidiacono JA. Bloom ET. Lipophilic statins suppress cytotoxicity by freshly isolated natural killer cells through modulation of granule exocytosis. International Immunology. 2007; 19(2):163-73. doi.org.10.1093/intimm/dxl133

19. Gullu S. Emral R. Bastemir M. Parkes AB. Lazarus JH. In vivo and in vitro effects of statins on lymphocytes in patients with Hashimoto’s thyroiditis. European Journal of Endocrinology. 2005; 153(1):41-8. doi.org.10.1530/eje.1.01941

20. Sirtori CR. The pharmacology of statins. Pharmacological Research. 2014; 88:3-11. doi.org.10.1016/j.phrs.2014.03.002

21. Kramer PA. Ravi S. Chacko B. Johnson MS. Darley-Usmar VM. A review of the mitochondrial and glycolytic metabolism in human platelets and leukocytes: implications for their use as bioenergetic biomarkers. Redox Biology. 2014; 2:206-10. doi.org.10.1016/j.redox.2013.12.026

22. Curry L. Almukhtar H. Alahmed J. Roberts R. Smith PA. Simvastatin inhibits L-type Ca2+-channel activity through impairment of mitochondrial function. Toxicological Sciences. 2019; 169(2):543-52. doi.org.10.1093/toxsci/kfz068

23. Bulhak AA. Gourine AV. Gonon AT. Sjöquist PO. Valen G. Pernow J. Oral pre‐treatment with rosuvastatin protects porcine myocardium from ischaemia/reperfusion injury via a mechanism related to nitric oxide but not to serum cholesterol level. Acta Physiologica Scandinavica. 2005;183(2):151-9. doi.org.10.1111/j.1365-201X.2004.01392.x

24. Severino A. Zara C. Campioni M. Flego D. Angelini G. Pedicino D. Giglio AF. Trotta F. Giubilato S. Pazzano V. Lucci C. Atorvastatin inhibits the immediate-early response gene EGR1 and improves the functional pro of CD4+ T-lymphocytes in acute coronary syndromes. Oncotarget. 2017; 8(11):17529. doi.org.10.18632/oncotarget.15420

25. Turcato G. Sanchis-Gomar F. Cervellin G. Zorzi E. Sivero V. Salvagno GL. Tenci A. Lippi G. Evaluation of neutrophil-lymphocyte and platelet-lymphocyte ratios as predictors of 30-day mortality in patients hospitalized for an episode of acute decompensated heart failure. Journal of Medical Biochemistry. 2019; 38(4):452-60. doi.org. 10.2478/jomb-2018-0044

Received on 04.04.2021 Modified on 05.06.2021

Research J. Pharm.and Tech 2022; 15(2):689-694.

DOI: 10.52711/0974-360X.2022.00114