MicroRNA155 Expression in Relation to BDCAF Scored Behçet's Disease in an Egyptian Patients' Sample
Sally S. Hassouna1, *, Manal Y. Tayel1, Dalal M. ElKaffash2, Ahmed M. Abdelhady3, Eman H. Elsayed1
1 Department of Internal Medicine, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Clinical and Chemical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
3 Department of Opthalmology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
To discover the possibility of using microRNA155 (miRNA155) expression level as a biomarker of Behçet's Disease (BD) activity or remission.
Thirty BD patients’ white blood cells (WBCs) miRNA155 expression was measured and compared to WBCs miRNA155 expression in 15 healthy subjects. Assessment of disease activity was done using Behçet's Disease Current Activity Form (BDCAF).
miRNA155 expression significantly decreases with the increase of BD activity scored by BDCAF.
Increased miRNA155 may be used as a biomarker of BD remission and thus in the disease follow up. There could be a prospect of treating the disease via microRNA 155 effect enhancement.
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
BD is a chronic multi-systemic disease characterized by oral ulcers more than 3 times in the year with at least two of the following: genital ulcers, cutaneous manifestations, ophthalmic, neurologic, or rheumatologic presentation [1Sakane T, Takeno M, Suzuki N, Inaba G. Behçet’s disease. N Engl J Med 1999; 341(17): 1284-91.[http://dx.doi.org/10.1056/NEJM199910213411707] [PMID: 10528040] ].
Pathogenesis remains unclear. However, both adaptive and innate immunity play a vital role, and multiple cytokines appeared to be engaged in different pathways of pathogenesis leading to tissue damage [2Lopalco G, Lucherini OM, Vitale A, Talarico R, Lopalco A, Galeazzi M, et al. Putative role of serum amyloid-A and proinflammatory cytokines as biomarkers for behcet’s disease.2015.]. A strong established relation between BD and human leukocytic antigen B51 (HLA-B*51) was evident in the last years [3Petrushkin H, Hasan MS, Stanford MR, Fortune F, Wallace GRBD. Behçet’s disease: Do natural killer cells play a significant role? Front Immunol 2015; 6: 134.[http://dx.doi.org/10.3389/fimmu.2015.00134] [PMID: 25852697] , 4Zierhut M, Mizuki N, Ohno S, et al. Immunology and functional genomics of Behçet’s disease. Cell Mol Life Sci 2003; 60(9): 1903-22.[http://dx.doi.org/10.1007/s00018-003-2333-3] [PMID: 14523551] ]. And there was an evidence of CD40 and CD40 ligands on peripheral blood lymphocytes and L- selectin expression on leucocytes of patients with BD [5Assaad-Khalil S, Abou Seif M, Elkaffash D. CD40 and CD40 ligands on peripheral blood lymphocytes in BD disease. 2006., 6Assaad-Khalil SH, Abou-Seif M, Youssef I, Farahat N. L-selectin expression on leukocytes of patients with Behçet’s disease. Adv Exp Med Biol 2003; 528: 273-8.[http://dx.doi.org/10.1007/0-306-48382-3_55] [PMID: 12918706] ]. Some biochemical markers were observed to correlate with BD activity for example, the level of Serum Amyloid A (SAA) which increase with the flares of ocular disease, neurological dysfunction, and the presence of oral ulcers, also it was suggested that increased levels of SAA might denote a thrombotic risk in the disease [7Vitale A, Rigante D, Lopalco G, et al. Serum amyloid-A in Behçet’s disease. Clin Rheumatol 2014; 33(8): 1165-7.[http://dx.doi.org/10.1007/s10067-014-2555-9] [PMID: 24659331] ]. Elevated cytokine levels of interleukin 6 (IL-6), interleukin 18 (IL-18), interleukin 8 (IL-8), and interferon α2a (IFN-α2a), and low CXCL11 levels were shown in BD. Also, it was observed that a signature of IL-6, and Tumor Necrosis Factor-α (TNF α) and T helper 17 (Th17) are present as well. This could contribute to uncover the role of cytokines in the appearance of specific clinical features of BD [2Lopalco G, Lucherini OM, Vitale A, Talarico R, Lopalco A, Galeazzi M, et al. Putative role of serum amyloid-A and proinflammatory cytokines as biomarkers for behcet’s disease.2015.].
MiRNAs are endogenous small naturally occurring double stranded (about 21-25 nucleotides) RNAs and were first described by Lee and colleagues in 1993 and were termed microRNAs in 2001. They play important regulatory roles in animals and plants through targeting mRNAs for suppression of translation or cleavage. Researchers have begun to understand that miRNAs influence the output of many protein-coding genes and are considered one of the gene regulatory molecules [8Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75(5): 843-54.[http://dx.doi.org/10.1016/0092-8674(93)90529-Y] [PMID: 8252621] ].
MicroRNAs exhibit a variable regulatory function in cell growth and differentiation, and are also associated with different human diseases. Previous research on miRNAs has revealed a new paradigm of gene regulations and pathways involved in the pathogenesis of autoimmune disorders, malignant and heart diseases [9O’Connell RM, Rao DS, Chaudhuri AA, Baltimore D. Physiological and pathological roles for microRNAs in the immune system. Nat Rev Immunol 2010; 10(2): 111-22.[http://dx.doi.org/10.1038/nri2708] [PMID: 20098459] ]. Thus human miRNAs are likely to be highly useful with a great promise as biomarkers in diseases’ diagnoses, and are targets for disease intervention and attractive for applying new therapies.
2. AIM OF THE STUDY
To show the correlation of miRNA155 expression with BD activity scored using BDCAF and the possibility of its usage as a disease activity or remission marker.
Seventeen patients had ocular involvement and 16 with non-ocular involvement were recruited from Alexandria university hospital, and out-patient clinics in a cross sectional pattern. Patients with a history of another auto-inflammatory or autoimmune disease, other form of vasculitis, hypercoagulable state, malignancy, chemotherapy, radiotherapy or long standing diabetes were excluded. An informed consent was taken from each patient. Three of the non-ocular diseased patients’ samples were excluded afterwards for technical errors.
Thirteen BD patients were having positive HLAB51, 18 patients were ANA positive and 7 had positive family history of BD.
Fifteen healthy subjects matched to the patients by age and sex served as the control for miRNA155 expression. Age of the patients ranged between 20 to 55 years old with a mean ± SD of 38.03± 9.94 years, and median of 36.5 years. No significant difference between the patients and control subjects. This was applied also for the sex.
Disease activity has been measured using “BDCAF” score depending on the presence or absence of mouth ulcers, genital ulcers erythema nodosum, pustules, arthralgia, arthritis, diarrhea, nausea/ vomiting, headaches, eye inflammation, new central nervous system involvement and new major vessel inflammation, (over the last four weeks before the visit) and accordingly the patients were divided into two groups; a higher disease activity group with a score equals to or more than 4 out of 12, and a lower disease activity group with a score less than 4. Patients included: are shown in Table 1.
Table 1 Clinical Manifestations in Patients that were Active Through the Four weeks Preceding the Study According to BDCAF. (Each of the Mentioned Items Counts for 1 Point as a Score of Disease Activity).
MiRNA155 in the group of highly active BD patients as scored by BDCAF ranged between 0.01 to 1.76 with a mean ± SD = 0.48 ± 0.53 and a median of 0.38, while in the lower activity group ranged between 0.01 to 6.56 with a mean ± SD = 2.08 ± 2.43 and a median of 0.73 and its expression in the control group ranged between 0.11 to 4.8 with a mean= 1.52 ± 1.27 and a median of 1.04. There was a significant difference between the higher activity patients and control group (p= 0.032), while there was no significant difference between lower activity patients and control group nor between higher activity and lower activity patients’ groups; p values were 1.00 and 0.127 respectively. There was no significant difference in miRNA155 expression in the whole patients group with a range extending from 0.01 – 6.56 with a mean ± SD = 1.28 ± 1.91 and a median 0.44 and control subject where (p = 0.075), the comparison is shown in Fig. (1).
Comparison between miRNA155 expression in the higher disease activity, lower disease activity and control groups.
Correlation between miRNA155 with BDCAF activity score in BD patients; a significant negative correlation between BDCAF score and miRNA155 expression level in BD patients involved in the study, with rs = -0.389, p = 0.034, this relation is shown in Fig. (2).
Correlation between miRNA155 expression in BD patients with activity of the disease scored using BDCAF.
BD is considered a serious syndrome that can lead to severe morbidity and sometimes mortality and unfortunately with no dependent biomarkers to assess activity or remission.
The study was made to show the relation between miRNA155 expression and disease activity.
The results showed a significant inversely proportional relation between miRNA155 expression in the WBCs and disease activity and so the increase of miRNA155 level denotes a disease remission.
This can be explained by knowing that several studies presented miR-155 as a major player in Dendritic Cell (DC) and B cell function and responses as what was discovered in [14Martinez-Nunez RT, Louafi F, Friedmann PS, Sanchez-Elsner T. MicroRNA-155 modulates the pathogen binding ability of dendritic cells (DCs) by down-regulation of DC-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). J Biol Chem 2009; 284(24): 16334-42.[http://dx.doi.org/10.1074/jbc.M109.011601] [PMID: 19386588] -16Ceppi M, Pereira PM, Dunand-Sauthier I, et al. MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells. Proc Natl Acad Sci USA 2009; 106(8): 2735-40.[http://dx.doi.org/10.1073/pnas.0811073106] [PMID: 19193853] ]. Two researches were conducted on mice lacking miR-155 showing aberrant B and T cell functions and defect in antigen presenting cells [17Rodriguez A, Vigorito E, Clare S, et al. Requirement of bic/microRNA-155 for normal immune function. Science 2007; 316(5824): 608-11.[http://dx.doi.org/10.1126/science.1139253] [PMID: 17463290] , 18Thai TH, Calado DP, Casola S, et al. Regulation of the germinal center response by microRNA-155. Science 2007; 316(5824): 604-8.[http://dx.doi.org/10.1126/science.1141229] [PMID: 17463289] ].
Also, the increased miRNA155 expression in disease remission may be owing to the fact mentioned in Martinez-Nunez et al, 2011 study [19Martinez-Nunez RT, Louafi F, Sanchez-Elsner T. The interleukin 13 (IL-13) pathway in human macrophages is modulated by microRNA-155 via direct targeting of interleukin 13 receptor alpha1 (IL13Ralpha1). J Biol Chem 2011; 286(3): 1786-94.[http://dx.doi.org/10.1074/jbc.M110.169367] [PMID: 21097505] ] about miRNA 155 implication in the inflammatory pro-Th1/M1 and that miRNA155 may be regulating the macrophages M1/M2 balance by modulation of IL13 effects.
It was said that there is an effect E26 transformation-specific-1 (Ets-1) on increased miRNA155 as Quinn et al., 2014 [20Quinn SR, Mangan NE, Caffrey BE, et al. The role of Ets2 transcription factor in the induction of microRNA-155 (miR-155) by lipopolysaccharide and its targeting by interleukin-10. J Biol Chem 2014; 289(7): 4316-25.[http://dx.doi.org/10.1074/jbc.M113.522730] [PMID: 24362029] ] said, and that Ets-1 is known to be a suppressor of T helper 17 suppressing the release of pathogenic interleukin 17 (IL17) as appeared in Moisan et al,. 2007 study [21Moisan J, Grenningloh R, Bettelli E, Oukka M, Ho IC. Ets-1 is a negative regulator of Th17 differentiation. J Exp Med 2007; 204(12): 2825-35.[http://dx.doi.org/10.1084/jem.20070994] [PMID: 17967903] ] IL-17 is increased in BD patients and there is a further increase with activity, ocular, and neurological involvement as what was said in Lopalco 2017 et al. [22Lopalco G, Lucherini OM, Lopalco A, et al. Cytokine signatures in mucocutaneous and ocular behçet’s disease. Front Immunol 2017; 8: 200.[http://dx.doi.org/10.3389/fimmu.2017.00200] [PMID: 28289419] ], and Dina et al., 2015 [23Al-Zifzaf DS, Mokbel AN, Abdelaziz DM. Interleukin-17 in Behçet's disease: Relation with clinical picture and disease activity 2015; 42(1): 34-8.].
Although our study included a more wide disease clinical spectrum, this finding of decreased miRNA155 with increased disease activity was shown before in ocular BD through Zhou et al., 2012 [24Zhou Q, Xiao X, Wang C, et al. Decreased microRNA-155 expression in ocular Behcet’s disease but not in Vogt Koyanagi Harada syndrome. Invest Ophthalmol Vis Sci 2012; 53(9): 5665-74.[http://dx.doi.org/10.1167/iovs.12-9832] [PMID: 22815348] ] which stated that miRNA155 expression levels in PBMCs and DCs was decreased in BD patients suffering from active uveitis and overexpression of miRNA155 in DCs increased the inhibitory interleukin 10 (Il 10) and decreased Il6 and Interleukin 1B (Il1B) expression which are pro-inflammatory cytokines which may contribute to disease flare. it was shown in some studies, such as Lopalco et al., 2015, that there is an important role Il6 in BD pathogenesis [2Lopalco G, Lucherini OM, Vitale A, Talarico R, Lopalco A, Galeazzi M, et al. Putative role of serum amyloid-A and proinflammatory cytokines as biomarkers for behcet’s disease.2015.].
Some other studies showed an opposite result to ours and that miRNA155 expression is increased in BD activity, as the study of Tili et al., 2007 which found that miR-155 is a potent pro-inflammatory microRNA and stated that Eμ-transgenic miR-155 mice have excessive uncontrolled inflammatory responses, giving excessive Tumor Necrosis Factor Alpha (TNFα) and septic shock hypersensitivity [25Tili E, Michaille JJ, Cimino A, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-α stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol 2007; 179(8): 5082-9.[http://dx.doi.org/10.4049/jimmunol.179.8.5082] [PMID: 17911593] ].
Other researches showed that miR-155 targeting Ets-1 regulates Th17 response and suppression of miR-155 decreases pathogenic IL-17-expressing T cells amount as in Na et al., 2016 study [26Na SY. Park MJ1, Park S, Lee ES. MicroRNA-155 regulates the Th17 immune response by targeting Ets-1 in BD disease. Clin Exp Rheumatol 2016; 34: S56-s63.[PMID: 27156371] ].
Also, Ryan et al. 2011 study found that miRNA155 fosters autoimmunity through inflammatory T cells development [27O’Connell RM, Kahn D, Gibson WS, et al. MicroRNA-155 promotes autoimmune inflammation by enhancing inflammatory T cell development. Immunity 2010; 33(4): 607-19.[http://dx.doi.org/10.1016/j.immuni.2010.09.009] [PMID: 20888269] ]. Also, it was mentioned in Rajasaki et al. 2017 that Resolvin D1, through inhibiting miRNA155, decreases experimental corneal immunopathology inflammation [28Rajasagi NK, Bhela S, Varanasi SK, Rouse BT. Frontline Science: Aspirin-triggered resolvin D1 controls herpes simplex virus-induced corneal immunopathology. J Leukoc Biol 2017; 102(5): 1159-71.[http://dx.doi.org/10.1189/jlb.3HI1216-511RR] [PMID: 28584076] ]. And in Zheng et al. 2012 and Chinenov et al., 2014, it was shown that glucocorticoids are effective in inflammation termination, mediated by miR-155 expression inhibition in a nuclear factor kappa-light-chain-enhancer of activated B cells and glucocorticoids receptor dependent way [29Zheng Y, Xiong S, Jiang P, et al. Glucocorticoids inhibit lipopolysaccharide-mediated inflammatory response by downregulating microRNA-155: A novel anti-inflammation mechanism. Free Radic Biol Med 2012; 52(8): 1307-17.[http://dx.doi.org/10.1016/j.freeradbiomed.2012.01.031] [PMID: 22326887] , 30Chinenov Y, Coppo M, Gupte R, Sacta MA, Rogatsky I. Glucocorticoid receptor coordinates transcription factor-dominated regulatory network in macrophages. BMC Genomics 2014; 15: 656.[http://dx.doi.org/10.1186/1471-2164-15-656] [PMID: 25099603] ].
The findings in some studies are consistent with ours and some are not, this is may be owing to different ethnicities, different disease activity in the patients in one study from another, also some researches which had the same results of ours or the opposite were depending only on animal studies and we should take this into consideration. Also, BDCAF scoring is almost subjective, depending on patients’ own words in many instances and this may be not so much accurate.
And thus miRNA155 role in BD pathogenesis is still controversial.
Studies on miRNA155 expression levels in active BD disease should be done on a large base to know if there is a cut off to say the disease is active or in remission and for confirmation of the possibility of its usage as a biomarker for follow up of the patient, and to do studies for Ets-1 enhancement or other means of miRNA155 promotion to see the effect on disease progression and potential of this for disease treatment.
ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Approval of Ethics Committee was obtained from the Local Ethics Committee of Alexandria University, with a serial number
020850, Chairperson: Dr. Maha Ghanem, IRB No. 0007555-FWA, No. 0018699, on 29-6-2016.
HUMAN AND ANIMAL RIGHTS
No Animals were used in this research. All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2013.
CONSENT FOR PUBLICATION
Written informed consent was taken from all the patients when they were enrolled.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
It is an onus to thank, All the patients and healthy volunteers who participated in this work, Dr. Eman Tayae, the lecturer of Clinical pathology at Alexandria University for her experience and cooperation in lab work, Dr. Rowayda M. Amin, the assistant lecturer of Opthalmology department at Alexandria University for her help in recruitment of many patients from the ophthalmology out-patient clinic and for her solicitude and care. Dr. Alya Elkayal and Dr. Asmaa Nasr, the clinical pathology residents for helping in doing many steps in the procedure, and the lab technicians for being keen and meticulous in their work.
Ceppi M, Pereira PM, Dunand-Sauthier I, et al. MicroRNA-155 modulates the interleukin-1 signaling pathway in activated human monocyte-derived dendritic cells. Proc Natl Acad Sci USA 2009; 106(8): 2735-40.[http://dx.doi.org/10.1073/pnas.0811073106] [PMID: 19193853]
Martinez-Nunez RT, Louafi F, Sanchez-Elsner T. The interleukin 13 (IL-13) pathway in human macrophages is modulated by microRNA-155 via direct targeting of interleukin 13 receptor alpha1 (IL13Ralpha1). J Biol Chem 2011; 286(3): 1786-94.[http://dx.doi.org/10.1074/jbc.M110.169367] [PMID: 21097505]
Quinn SR, Mangan NE, Caffrey BE, et al. The role of Ets2 transcription factor in the induction of microRNA-155 (miR-155) by lipopolysaccharide and its targeting by interleukin-10. J Biol Chem 2014; 289(7): 4316-25.[http://dx.doi.org/10.1074/jbc.M113.522730] [PMID: 24362029]
Al-Zifzaf DS, Mokbel AN, Abdelaziz DM. Interleukin-17 in Behçet's disease: Relation with clinical picture and disease activity 2015; 42(1): 34-8.
Zhou Q, Xiao X, Wang C, et al. Decreased microRNA-155 expression in ocular Behcet’s disease but not in Vogt Koyanagi Harada syndrome. Invest Ophthalmol Vis Sci 2012; 53(9): 5665-74.[http://dx.doi.org/10.1167/iovs.12-9832] [PMID: 22815348]
Tili E, Michaille JJ, Cimino A, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-α stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol 2007; 179(8): 5082-9.[http://dx.doi.org/10.4049/jimmunol.179.8.5082] [PMID: 17911593]
Na SY. Park MJ1, Park S, Lee ES. MicroRNA-155 regulates the Th17 immune response by targeting Ets-1 in BD disease. Clin Exp Rheumatol 2016; 34: S56-s63.[PMID: 27156371]