Regulation of CD4+CD25+FOXP3+ Treg Cells in Systemic Lupus Erythematosus (SLE): Association With miRNAs Expression

Various genetic factors are controlling regulatory cells T (Treg) cell function, such as miRNAs. Interfering in the miRNA synthesis pathway in Treg cells could result in loss of Tregs' regulatory function, leading to the promotion of inammatory settings and autoimmunity. This study was designed to investigate the role of miRNA in regulating Treg cells in SLE patients. Treg's frequency was determined using ow cytometry in 100 SLE patients’ and100 healthy controls. Expression of miR-21, miR-24, miR125, miR-146a, miR-148a, and miR-155 was estimated in peripheral blood mononuclear cells (PBMCs) using quantitative real-time polymerase chain reaction (qRT-PCR). The ROC curve evaluated the diagnostic role of miRNAs in SLE. A signicant elevation (p<0.001) in Treg cells in SLE patients than controls was observed, with a maximum increase inactive SLE cases. SLE patients exhibit a signicant increase in miR-21 (p<0.01), miR-148a (p<0.001), miR-146a (p<0.05) and miR-155 (p<0.001) and signicant reduction in miR-24 (p<0.001). An insignicant decrease in miR-125 was observed in SLE patients. The best sensitivity and specicity were detected in miR-148a (88%, 70%) at a cutoff value of 1. 065. Tregs were positively correlated with miR-21(r=0.333, p<0.05), miR-146a (r=0.589, p<0.01) and miR-148a (r=0.309, p<0.05). In conclusion, this research provides a piece of novel information regarding Treg cells' in SLE patients. Our results pointed to the substantial role of miRNAs in controlling Treg cells in lupus. To validate our interesting results, more researches are needed.


Introduction
Systemic lupus erythematosus (SLE) is a serious autoimmune multisystem disorder characterized by a lack of self-antigen immune tolerance, resulting in the continuous development of pathogenic autoantibodies, lymphocyte activation, and release of in ammatory mediators [1][2][3].The subsequent production of autoantibodies by autoreactive B cells is one of the main pathological factors in SLE, contributing to the production and deposition of immune-complexes leading to tissue damage [4][5][6].Both pathogenesis and autoantibody formation are dependent upon CD4 + T cells [7].Based on cytokine patterns, naive CD4 + T cells can be divided into multiple subsets, including Th1, Th2, Th17, and Regulatory T cells (Treg) [7,8].
Tregs (CD4 + CD25 + FOXP3 + ) cells are a specialized type of T cells that suppress the immune response, thereby maintaining homeostasis and self-tolerance [9]. It plays a vital role in the tolerance induction and sanctuary against autoimmunity [11,12].Treg cells regulate the in ammatory activity [12,13]by suppressing the effector T cells and inducing the release of antiin ammatory as well as tissue repair cytokines [e.g., transforming growth factor-β (TGF-β),interleukin-10 (IL-10) and IL -35]. TGF-β, working together with IL-10 to induce Treg differentiation from naïve T cells [14]. The equilibrium between the effector and regulatory T cells determines whether an autoimmune response can be triggered and propagated by autoreactive cells or not [12]. While a great deal of effort has been made to shed some light on the Treg imbalance in SLE, contradictory results have been shown [15][16][17][18][19][20][21][22][23][24]. epigenetic, that coordinate the possible divergence of immune cells in lupus, our work performed a ow cytometric analysis of CD4 + CD25 + FOXP3 + Treg cells in SLE patients at various stages of disease activity. The potential impact of multiple expression pro les of microRNAs (miR-21, -24, -125, -146a, -148a, and − 155) on Treg cells was also examined.

SLE patients and healthy controls
Our study included 100consecutive patients who met the American College of Rheumatology (ACR) criteria for diagnosing SLE [33,34]. They were outpatients at the Rheumatology Department at El-Eini Hospitals, Cairo University, Egypt. The mean duration of SLE was 6.97 ± 5.73 years. Disease activity was assessed for all the lupus patients on the day of blood sampling by the SLE disease activity index (SLEDAI), and they were divided into active (SLEDAI score ≥ 6) and inactive (a SLEDAI score of < 6) [35]. The exclusion was made for patients with concomitant malignant diseases, infections, diabetes, abnormal lipid pro le, and pregnant women. One hundred participants matched by age and sex were enrolled as a normal control group with no history of autoimmune disorders or immunosuppressive drug treatment. Through a standardized interview and physical examination, demographic and clinical characteristics were collected. The local Ethics Committee of Cairo University con rmed the study plan. Both patients and healthy controls agreed to participate in this research, and all had received informed consent.
Based on their clinical status, SLE patients were divided into groups with active or inactive organ involvement. Patients were divided based on clinical manifestation into SLE patients with: skin involvement (lupus rash), photosensitivity (discoid lupus); active joint involvement (arthritis with synovial swelling), active hematologic involvement (thrombocytopenia, lymphocytopenia, or leukocytopenia or hemolytic anemia); active renal involvement (nephritis with proteinuria > 0.5 g protein/24 h and/or active nephritic sediment).

Flow cytometric detection of T-lymphocytes
In sterile ethylenediaminetetraacetic acid (EDTA) tubes, 5 ml of venous blood were withdrawn. Ficoll-Hypaque separating media (Biowest SAS, Nuaillé, France) was used to isolate human peripheral blood mononuclear cells (PBMCs) from the blood, and Treg cells were identi ed using multiple staining's with three monoclonal antibodies against CD4, CD25, and FOXP3 as previously described [36, 37].
2.2. RNA extraction and quanti cation of miRNA expression levels RNA was extracted from PBMCs of all participants using a TRIzol-based miRNA isolation kit (Life Technologies Ltd. UK)as described in the manufacturer's instructions. The purity and concentration of RNA were spectrophotometrically assessed by NanoDrop™ 2000/2000c (Thermo Fisher Scienti c, Waltham, MA, USA). The RNA integrity was checked by 1%agarose gel electrophoresis. miRNAs expression was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). miScriptII RT Transcription Kit (QIAGEN Valencia, CA, United States) is used to transcribe 100 ng RNA from each sample to cDNA. The reaction was performed at 37ºC for 60 min, followed by incubation at 95ºC for 5 min to inactivate the enzyme. Expression of Hs_miR-21, Hs_miR-24, Hs_miR-125a, Hs_miR-146a, Hs_miR-148a, and Hs_miR-155 was measured using miScript SYBR Green PCR Kit (Qiagen) and miScript Primer Assays (Qiagen) according to the supplemented protocol. The PCR cycling conditions were performed in AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA) as follow: 95ºC for 15 min for initial denaturation, followed by 40 cycles at 95°C for 15 s for denaturation, 55°C for 30 s for annealing and 72°C for 30 s for an extension, and a nal stage were 95ºC for 15 s, 60ºC for 1 min and 95ºC for 15s.As endogenous housekeeping control, SNORD68 and U6B small nuclear RNA (RNU6B) expression were used for data normalization. All calculations of miRNA expression levels were done as previously reported by El-Maadawy et al. [37].
2.3. Statistical analysis SPSS 21.0 was used for all statistical analysis. Where applicable, data were statistically de ned in terms of mean, standard error (SE), or frequencies. The Student's T-test for statistical analysis of parametric data and the Mann-Whitney U test for non-parametric data were used. Comparisons between groups were made using a one-way analysis of variance (ANOVA).By a receiver operating characteristic (ROC) curve, sensitivity versus the false positive frequency (one-speci city) for miRNAs was analyzed. Person's or Spearman's correlation test was used to assessing the correlation between variables. All twosided values with a P-value of less than 0.05 were considered. signi cant.

Demographic, biochemical, and clinical characteristic of SLE patients
The present study was conducted on 100 SLE patients; 87 women and 13 men. Their mean age was 32.8 ± 1.0 years. One hundred healthy individuals were run in parallel (85 females and 15 males, with mean age 27.22 ± 7.90). All patients and controls were examined for the biochemical parameters associated with the disease.   (Fig. 2). Our results showed a signi cant increase in active and inactive patients (p < 0.001 and p < 0.05, respectively) compared to healthy controls with a maximum elevation in inactive ones. Although active SLE patients have more Treg than inactive patients, this increase is statistically insigni cant.

miRNAs expression levels in SLE
As an overview of the whole lupus patients, our data showed a signi cant diminution in the expression ofmiR-24 in SLE patients compared to healthy controls (p < 0.001). miR-125 was also decreased in SLE patients but insigni cantly. On the other side, miR-21, miR-146a, miR-148a and miR-155 were signi cantly elevated (p < 0.01, p < 0.05, p < 0.001andp < 0.001; respectively) in lupus patients in relation to normal controls. Concerning the activity of the disease, our data showed a reduction in miR-24 and miR-125 and elevation in miR-21, miR-146a, miR-148a, and miR-155 in both groups (Fig. 3) as compared with normal controls. Regarding active and inactive SLE patients, no signi cant changes were found in the expression of miRNAs (miR-24 and miR-125 and elevation in miR-21, miR-146a, miR-148a, and miR-155) between both groups.
To distinguish between SLE patients and healthy control groups, we used ROC curve analysis to estimate the cutoff value for all calculated miRNAs (Fig. 5).According to the ROC curve results, the highest value of the area under the curve (AUC) was found in miR-148a(0.806), followed by miR-155 (0.764), and nally miR-21 (0.625).The highest levels of sensitivity and speci city were discovered ( Table 3). The best sensitivity and speci city were detected in miR-148a (88% and 70%; respectively) at a cutoff value of 1.065 (Table 3).

Association between SLE clinical manifestations and miRNAs and Tregs.
The association between SLE clinical disease manifestations and miRNAs secretion levels was demonstrated (Table 4). Patients with renal manifestations or neutropenia had signi cantly higher levels of miR-21 (p < 0.01and p < 0.05, respectively), while SLE patients with Raynauds phenomena had a signi cantly lower expression of miR-21 (p < 0.05) compared with those without these phenomena. SLE patients with serositis or renal disorder had signi cantly higher levels of miR-24 (p < 0.001and p < 0.05, respectively) compared to patients without these manifestations. Vasculitis manifestation was accompanied by a signi cant elevation (p < 0.01) of miR-125. Signi cantly elevated levels of miR-146a were reported in SLE patients with photosensitivity or lymphopenia (p < 0.05). Lupus patients with neutropenia had signi cantly higher levels of miR-155 than SLE patients without this manifestation (p < 0.05). We found a remarkable observation in miR-148a, which is signi cantly elevated (p < 0.01) in all hematological abnormalities (leucopenia, neutropenia, or lymphopenia) in addition to vasculitis (p < 0.01), serositis (p < 0.01), or renal manifestations (p < 0.01).
According to the type of treatment (Table 5), SLE patients treated with Endoxan, Imuran, and biologic had signi cantly higher levels (p < 0.01) of miR-21 compared to SLE patients without these treatments. A reduction in miR-125 (p < 0.001), miR-146a (p < 0.01), and miR-155 (p < 0.05) was observed in response to the treatment with Endoxan, Imuran, and Biologic.
A signi cant increase in Treg cells was observed in SLE patients with photosensitivity (p < 0.01) and with Raynauds (p < 0.001) as compared with patients without these manifestations (Table 6). Alternatively, Treg cells didn't show any statistical signi cance with the other clinical manifestations of the disease or any treatment regimens.

Discussion
Tregs suppress self-reactive T cells and inhibit their number and functions, helping to maintain peripheral tolerance and prevent the onset of autoimmune diseases [38,39]. Tregs suppress the functions of a number of cell types, including CD4 + TH cells, B cells, CD8 + cytotoxic T lymphocytes (CTLs), and antigen-presenting cells(APC), to effectively block immune responses, in ammation, and tissue destruction [40][41][42].In the present study, alteration in Treg cells' frequency in SLE has been analyzed in the light of miRNAs expression, which possibly in uences Tregs.
Our study observed a substantial increase in Treg cells in both active and inactive SLE patients compared to controls, with the most signi cant increase in active ones. This result agreed with Singla et al.'s(2017) [43] results, who reported a signi cant increase in Tregs in childhood SLE and mentioned that active lupus patients had a higher percentage than inactive lupus patients do. A previous study by Suarezet al. (2006) [44] also observed a signi cant elevation in both active and inactive SLE patients with a maximum increase in inactive ones. In contrast,  [45]found an insigni cant difference in Treg cells' percentage in SLE patients. Knowing that corticosteroids (glucocorticoids and cyclophosphamide) were used as a regular treatment of lupus due to their suppressing effect on the immune response, speci cally the development of pro-in ammatory cytokines, the observed rise in Tregs in our SLE patient might be returned to the impact of immunosuppressive therapies. These therapeutics have been proven to augment Tregs frequency in several conditions, including lupus [46][47][48][49].
Several miRNAs have been discovered to be essential in immune homeostasis. The role of microRNAs in immune cell lineage differentiation and their physiological functions in maintaining normal innate and adaptive responses is well known [50,51]. Aberrations in the miRNA-mediated immune-cell development and function regulation have been related to autoimmune diseases [52][53][54]. Intuitively, miRNA dysregulation is one of the main contributors to the collapse of selftolerance, leading to autoimmunity [55].
In the current study, there was a lowering in miR-125a expression level in both active and inactive phases of the disease compared to the healthy group. These ndings agree with Zhaoet al. (2010) [56] and Wang et al. (2012) [57], who reported a reduction in miR-125 level in SLE patients. In T cells isolated from lupus patients, diminished levels of miR-125a had been reported [58]. miR-125a promotes the up-regulation of the in ammatory chemokine RANTES, which is needed for the adverse effects of in ammatory processes. Its de ciency impairs Treg maintenance and immunoregulatory capacity, while over expression of miR-125a stabilizes Treg-mediated self-tolerance [59].  [57], who reported an unexpected reduction in the expression level of miR-155 in SLE patients. Over expression of miR-155 contributes to the development of antibodies, irregular T cell differentiation, kidney failure, and lupus-like symptoms [69,70]. Some miRNAs, such as miR-155, commonly associated with a compromised immune response and increases disease activity, were differentially expressed in multiple autoimmune diseases [71].
We demonstrated a signi cant increase in miR-21 was observed in both active and inactive status of the disease compared to healthy controls. In accordance with this data, the study of Wanget al.  [72], who observed a signi cant increase in miR-21.In accordance, patients with active disease have substantially higher levels of miR-21 in their PBMC than normal subjects and patients with inactive disease [73]. Elevated miR-21 levels promoted CD4 + T cell activation, B cell hyper-responsiveness, and over expression of autoimmune-associated methylation-sensitive genes through repression of DNMT1, PDCD4, or PTEN expression [72,74,75]. Besides, the inhibition of miR-21 in CD4 + T cells from SLE patients might reverse T cells' activation [74,76].  [72], who observed that miR-148a was up-regulated in SLE patients. miR-148a expression was up-regulated in CD4 + T cells from patients with SLE patient. miR-21, miR-126, and miR-148a over-expression resulted in DNA hypomethylation in CD4 + T cells by direct inhibition of DNMT1 protein expression, thus inducing CD4 + T cell activation and secretion of autoimmune-related proteins, such as CD70, CD11a, and LFA-1 [72,77,76].In females, DNA methylation serves as a housekeeping mechanism for physiological X-chromosome inactivation [78][79][80][81].It might be estimated that increased circulating miR-21 and miR-148a, in turn, might also accelerate disease progression through the cell-cell communication processes between these apoptotic bodies, exosomes, and target cells, such as quiescent lymphocytes [76]. Zhang et al. (2020) [71] pointed to the elevation of miR-148a, which is generally associated with the immune response and increases the disease's activity.
In conclusion, we approved the numeric rising in Treg cells' frequency in SLE patients, especially those in an active state. Although, we stressed the idea that these elevated cells might be malfunctioning. Studying the expression of some miRNAs associated with Treg cells pointed to the increase in miR146a, miR155 miR148a, and miR-21, coinciding with the reduction of miR-24.We hypothesized that the increase in miR-21, miR-148a, and miR-155 (Treg positive regulators) accompanied by a decrease of miR-24 (Treg negative regulators) favors the elevation of Treg cells, leading to this observed increase of Treg cell frequency. There is a lack of consensus in the research on the relationship between Treg and rheumatic diseases. The majority of evidence proposes Treg cells' impairment, quantitatively and/or qualitatively. Herein, our results provide a novel insight into Treg-miRNA's role in lupus patients' regulation network.
However, our study has some potential limitations, such as the lack of previous studies on some miRNAs in SLE patients (such as miR-24) and using a sorter to test the function of detected Treg cells. Thus, further studies are needed to con rm our ndings. We performed the same research on another important autoimmune disease (RA) to examine our assumption's strength in view of this hope.

Declarations
Con ict of interest: The authors declare that they have no con ict of interest.      Figure 1 Flow cytometric detection of Treg cells The Roc curve of miRNAs in SLE patients