Regulatory T cells and Disease damage in systemic lupus erythematosus

2000 (SLEDAI -2k) while disease damage was measured using the Systemic Lupus international

https://ejmr.journals.ekb.eg/2000 (SLEDAI -2k) while disease damage was measured using the Systemic Lupus international collaborating clinics American College of Rheumatology Damage Index (SLICC/ACR DI) which were correlated with T regulatory cells percentage in cases only.Results: Treg cells percentage was significantly decreased in SLE patients when compared with healthy controls (0.1% to 0.9% vs 0.9% to 2.1%) (p< 0.001).As regard SLEDAI -2k, there was a negative correlation between Treg cells percentile and SLEDAI-2k (p< 0.001).Also there was a negative correlation between Treg cells percentile and damage index (SLICC/ACR DI) (p< 0.001).Regarding the correlation between SLEDAI -2K with damage index (SLICC/ACR DI), we found highly significant positive correlation (p< 0.001).Conclusion: Our study showed that Tregs percentile were significantly lower in SLE patients when compared with healthy controls.Treg cells % have significant association with SLEDAI and damage index suggesting the value of Tregs as activity biomarker and marker of damage.[5]The ability of Foxp3+   The confidence interval in probability (Pvalue) was set to 95% and the margin of error accepted was set to 5%.So, the p-value was considered significant as the following: Probability (P-value) < 0.05 was considered significant, P < 0.001 was considered as highly significant and P > 0.05 was considered insignificant.

Declaration of conflicting interests
The Systemic lupus erythematosus is a multiorgan autoimmune disease with diverse and varied clinical manifestations and longterm outcomes with exacerbations and remissions.Irreversible organ damage is a primary outcome in SLE.It is occured during the course of SLE caused by both the disease itself and therapies received by patients.Damage to tissues and organs is linked to a higher chance of mortality.[1] CD4+CD25+Foxp3+ Treg cells are a type of CD4+ cell that suppresses the immune response and promotes self-tolerance.Treg cells are mainly produced in the thymus via self-antigen recognition, although they can also be found in the peripheral lymphoid organs.[2], [3]Tregs express high levels of IL-2 receptor alpha (CD25) and the transcription factor forkhead box P3 (Foxp3).Alongside IL-2 and CD25, which facilitate https://ejmr.journals.ekb.eg/ the development, function and stability of Tregs, numerous other cell-surface receptors for Tregs have been identified.IL-2 supports Treg development in the thymus and is also needed for their survival and function in the periphery.[4]The forkhead winged-helix transcription factor Foxp3 (forkhead box p3) discovery as master regulator for Treg cells added a key marker for this T cell subset.Foxp3, in fact, is constitutively expressed at high levels in both natural and adaptive CD4+CD25high Treg cells in human beings and mice.It is required for the natural Treg lineage commitment in the thymus and is essential in stabilizing and amplifying a Treg program induced by interaction between Treg precursors and stromal cells in the thymus.

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SLICC/ACR DI) Damage Index [11].The cut-off for organ damage was SLICC 0 =no organ damage, SLICC 1-3 =moderate organ damage, and SLICC >3 = severe organ damage.Following these processes, all participants underwent a comprehensive medical history, clinical examination, and flowcytometric measurement of peripheral Treg cells using anti CD4, anti CD25, and anti FOXP3 monoclonal antibodies.Human peripheral blood mononuclear cells (PBMCs) were freshly separated by Ficoll density gradient centrifugation then washed with 2.0 mL of flowcytometry staining buffer (R&D Systems®) by spinning at 300 x g for 5 minutes, using 5.0 mL flowcytometry tubes.After that 10 μL of CD4-FITC and 10 μL of CD25-APC antibodies or isotype controls (R&D Systems®) were added which incubated for 30-45 minutes at 2-8 °C in the https://ejmr.journals.ekb.eg/dark.The cells were washed two times with cold 1X Phosphate buffer saline (PBS) then resuspended in 0.5 ml of fresh 1X FoxP3/Transcription Factor Fixation Buffer (R&D Systems®) and incubated at 2-8 °C for 30 minutes.During this incubation, 1X FoxP3/Transcription Factor Permeabilization and Wash Buffer was maked up by diluting FoxP3/Transcription Factor Permeabilization and then Buffer (10X) was washed with distilled water (i.e. 100 μL FoxP3/Transcription Factor Permeabilization and the Washed Buffer (10X) + 900 μL diH20) were kept at 2-8 °C.The mixture was washed two times with fresh, cold, 1X FoxP3 Permeabilization and Wash Buffer.10 μL of FoxP3 antibody or the rabbit IgG-PE isotype control included in the kit was added to the cells and incubated for 30 minutes at 2-8 °C.Then the cells were washed one time with cold 1X FoxP3 Permeabilization and Wash Buffer.The cells were resuspended in Flowcytometry Staining Buffer and run on a flowcytometer.The data was collected and analysed on an EPICS XL flowcytometer with the SYSTEM II version3 software and a standard 3-color filter configuration.Lymphocytes were gated using forward and side scatter properties and CD4+ cells were identified using CD4 expression.CD25high CD4+ Tcells were distinguished from CD25dim CD4+ Tcells after the gated CD4+ Tcells were tested for both CD25 expressions usingPE-anti-Foxp3.Finally, FoxP3 expression was evaluated in CD25highCD4+ Tcells .Treg cells were expressed as a percent of CD4+ T cells as shown in figure 1.

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Systemic lupus erythematosus is a complex autoimmune disease that has a chronic relapsing-remitting course with a wide range of symptoms, ranging from moderate to lifethreatening sickness[12].The current work makes a scientific addition by estimating the value of CD4+ CD25+FoxP3+ Treg cells as a biomarker of activity and damage.Progress in understanding the immunopathogenesis of SLE could also lead to a better knowledge of the disease mechanism and tangible benefits for patients, making it feasible to give the https://ejmr.journals.ekb.eg/best treatment for lupus patients and decrease the damage effect of the disease.When compared to healthy controls, which range from 0.9 % to 2.1 %, Treg cells in the peripheral blood of systemic lupus patients (0.1 percent to 0.9 percent) were significantly lower.These results were in agreement withCai et al. (2012); Habibagahi et al. (2011); Henriques et al. (2010); Ma et al. (2013) and Xing et al. (2012) who investigated the frequencies of circulating Treg in SLE and found a significant decrease in circulating Treg in peripheral blood of diseased patients when compared with controls.The finding of reduced level of Tregs in SLE was explained by the decrease expression of CD25 in Tregs from patients with SLE linked with the production of IL-2 by memory T cells indicating that IL-2 deficiency in SLE patients reflects CD25 reduction in Tregs.Reduced expression of CD25 may impact Treg function in SLE patients because IL-2 receptor-dependent activation of transcription factor STAT5 is required for the suppressive function of Tregs [18].Yates et al. (2008) observed that the number of circulating CD4+Tregs expressing CD25 molecules and Foxp3 in lupus patients remained stable.Azab et al. (2008) and Yan et al. (2008), on the other hand, have found a larger number of these cells in SLE patients.Differences in the use of specific phenotypic markers for identifying Tregs, as well as the methodology for isolating or stimulating these cells prior to staining, may have caused these inconsistencies.In contrast to El-Maraghy et al. (2018) and Bonelli et al. (2014), who found a positive correlation between haematological manifestations including thrombocytopenia and leucopenia, there was no statistically significant correlation between T regulatory cells percent and haematological indices.This could be explained by the overlap of organ manifestations, as most SLE patients suffer from haematological manifestations as well as renal involvement.In this study, there was a statistically significant negative correlation between the percentage of T regulatory cells and CRP and ESR.These findings are in agreement with those of M. Nabil et al. (2018) and Lee et al. (2008), who look at the relationship between Treg and ESR as an inflammatory marker, as ESR levels are used to assess disease relapse and distinguish infection.However,Atfy et al. (2009) and Barâth et al. (2007) discovered no statistically significant link between the percentages of these cells in lupus patients and ESR.The percentage of T regulatory cells and serum Creatinine had a negative relationship.This finding is consistent with Maher et al. (2019), who found that Treg has a role in https://ejmr.journals.ekb.eg/lupus nephritis.However, Atfy et al. (2009) and Barâth et al. (2007) observed no significant link between the percentages of these cells and creatinine levels in lupus patients.There was a statistically significant positive correlation between SLEDAI and ESR, which is consistent with Santhanamet al (2015).Mirzayan et al. (2000) and Chang et al. (2002) couldn't find any link between ESR and disease activity in their studies.Many complicating factors, such as anaemia, hypoalbuminemia, high cholesterol, or high gammaglobulin levels, might raise ESR.As a result, all of these confounding factors must be eliminated, and it would be ideal if ESR and its relationship with disease activity could be investigated throughout each flare episode.Damage index and creatinine level showed a positive significant correlation, confirming the findings of Ghazali et al. (2018), who investigated the relationship between damage index and other variables affecting lupus patients, demonstrating that renal affection is an irreversible disorder.SLEDAI and damage index have a significant positive correlation, which is consistent with the findings of Bruce et al. (2015), who investigated the relationship between damage index and several factors affecting patients with SLE, including older age at diagnosis, longer duration of SLE, African-Caribbean or Asian ethnicity, high disease activity at diagnosis, and greater overall activity duration.The fact that disease activity and damage have such a strong relationship suggests that they work together to accelerate the onset of permanent organ alterations.On correlating damage index with Treg %, we found significant negative correlation which to our knowledge has not been discussed before in other studies indicating the role of T regulatory cells in the pathogenesis of SLE and its decrease leads to irreversible damage.Conclusion: Treg cell percentage has a substantial relationship with SLEDAI and damage index, implying that Tregs can be used as an activity biomarker and damage marker.The value of Tregs as activity and damage biomarkers may help in assessing disease status in controversial circumstances and prevent further permanent damage.Further studies on larger scales are required for further assessment of role of Treg in SLE and more correlation with parameters affecting mortality and morbidity of the disease.

2. Material and Methods:
expression levels displayed by Treg cells.[7] In SLE, Qualitative and/or quantitative disorders affecting Treg cells may lead to the disequilibrium in peripheral tolerance and may participate in triggering the disease.[8] disease.[9] So the aim of the study was to assess the concentration of CD4+ CD25+ FoxP3+ natural Treg in SLE patients and study its relation to SLEDAI -2k activity index and damage index (SLICC/ACR DI).

regulatory cells% SLEDAI Damage index r-value p-value r- value p-value r-value p-value
Table (4): Correlation between T regulatory cells% with SLEDAI and Damage index, using Pearson Correlation Coefficient in patients group.