The Association Between Regulatory T-Cells and T Helper 17 Cells with Blood Pressure in Hypertension

The Association Between Regulatory T-Cells and T Helper 17 Cells with Blood Pressure in Hypertension | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Association Between Regulatory T-Cells and T Helper 17 Cells with Blood Pressure in Hypertension Swastya Dwi Putra, Bambang Widyantoro, Dian Zamroni, Sisca Natalia Siagian, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6635766/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: The immune system is currently been postulated to play a role in hypertension. The imbalance of Regulatory T-cells and T helper-17 cells has been found to be the cause of hypertension in animal studies. Method and Results: This case-control study was conducted at the National Cardiovascular Center of Harapan Kita and Universitas Indonesia Hospital from August to December 2022. Subjects with essential hypertension and normotension who visited the outpatient clinic were consecutively included. A flow cytometry examination was performed to quantify Regulatory T-cell and T helper-17 cell levels in the blood. The study consisted of 40 subjects from each group. The results showed that Regulatory T-cells were lower in the hypertension group compared to the normotension group (p < 0.001). We observed a strong correlation between Regulatory T-cells and both systolic blood pressure (r = -0.733, p < 0.001) and diastolic blood pressure (r = -0.613, p < 0.001). T helper-17 cells were also higher in the hypertension group (p < 0.001). Furthermore, we identified a moderate correlation between T helper-17 cells and systolic blood pressure (r = 0.357, p < 0.001) and diastolic blood pressure (r = 0.242 and p = 0.015). The ratio of T helper-17 cells to Regulatory T-cells was significantly higher (p < 0.001) in subjects with hypertension. Conclusion: We revealed an association between Regulatory T-cells and T helper-17 cells in human hypertension. We further concluded that human hypertension is characterized by low Regulatory T-cells and high T helper-17 cells. Regulatory T-cells T helper-17 cells hypertension immune system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The discovery of autoantibodies in artery samples from “hypertensive cadavers” more than 50 years ago raised the possibility of a relationship between the immune system and hypertension. 1 Since then, various studies on the role of the immune system in hypertension have emerged. Animal studies revealed that an imbalance of the T helper-17 (Th17) cell to Regulatory T-cell (Treg) ratio plays a critical role in hypertension. 2 Treg was first discovered as a novel regulator of hypertension through a study conducted by Dr. Schiffrin’s team. Using consomic-modified Brown Norway rats of normotensive strain chromosome 2 with the Dahl salt-sensitive genome, which overexpresses Treg cells, they found lower blood pressure and vascular hypertrophy compared to Dahl salt-sensitive rats. The consomic rats have also increased production of the anti-inflammatory cytokines interleukin (IL)-10 and transforming growth factor (TGF)- . 3 Another study conducted by Barhoumi demonstrated the ability of Treg to inhibit angiotensin II-mediated vascular damage. In contrast, angiotensin II-induced a significant reduction of Treg in the renal cortex. 4 In concordance with Barhoumi’s findings, Kasat et al. also showed that Treg inhibits aldosterone-mediated vascular injury through the interaction between innate and adaptive immunity. 5 Moreover, Mian et al. showed that Treg insufficiency amplified angiotensin II-induced microvascular damage. 6 Furthermore, a human study by Ciuceis et al. revealed that several lymphocytes, including Treg, were associated with microvascular remodeling. 7 A recent study also showed the importance of Th17 cells in hypertension. A study conducted by Madhur et al. demonstrated a direct correlation between Th17 cells and angiotensin II (Ang II)-induced hypertension where they found preservation of vascular structure, decreased superoxide production, and low aortic T-cell infiltration in IL-17 null mice compared with wild-type. 8 Based on the emerging evidence of the Th17/Treg cell role in animal hypertension studies, this study investigated the association of those cells with human hypertension. MATERIALS AND METHODS 1.1. Study Design and Population This is a case-control study conducted from August 2022 to December 2022. All patients admitted to the outpatient clinic in the National Cardiovascular Center of Harapan Kita and Universitas Indonesia Hospital were screened for eligibility. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by the institution’s human research committee and board committee ethics. 1.2. Subject Recruitment We included all patients aged 18 years or older who were admitted to the outpatient clinic with essential hypertension or normotension. We excluded patients with diabetes mellitus, coronary heart disease, stroke, peripheral artery disease, heart failure, chronic kidney disease, infection, cancer, autoimmune disease, and statin therapy. Informed consent was obtained from each included patient. Each patient who was admitted to the outpatient clinic was screened with a history, physical examination, blood pressure measurement, waist circumference measurement, and body mass index assessment. Blood pressure was measured with a calibrated digital OMRON TM HEM 8712 using JNC 8 protocols. Body weight was measured with a calibrated Camry weighing scale using the standardized protocol. Height was measured with a standardized Microtoise. Waist circumference was measured with a measuring tape measure using the World Health Organization protocol. Selected subjects then underwent further examination, including a dietary habit intake interview and a blood examination. 1.3. Hypertension and Dietary Intake Determination Office blood pressure measurement was taken during the first meeting. Additionally, for each participant, home blood pressure measurements (HBPM) were conducted over a period of five days. Participants measured their blood pressure twice a day: in the morning when they woke up and at night when they were going to sleep. Blood pressure was measured with a calibrated digital OMRON TM HEM 8712 using JNC 8 protocols. The food frequency and 24-hour recall questionnaires were utilized for calculating dietary intake. The 24-hour recall was conducted by two trained enumerators who assisted interviewees with scripts, visuals, and other tools, including measuring cups and food models, while conducting interviews in Indonesian. The food preparation process, brand names (if applicable), and supplements utilized were all noted thoroughly. 1.4. Blood Sample Collection and Immunological Studies The blood suspension was collected in a vacutainer tube coated with EDTA. The blood was then processed for peripheral blood mononuclear cell (PBMC) isolation by density gradient centrifugation using Lymphoprep™ (StemCell Technologies). Blood was mixed with Lymphoprep™ in a 1:1 volume. The mixture was then centrifuged at 800 x g for 20 minutes at 20°C. The part of the buffy coat that was under the plasma was taken and resuspended with a complete PBMC medium for examination using flow cytometry. Flow cytometry examination was done using the FACS ARIA III (BD Bioscience) tool. For examining IL-17A and Foxp3 markers, staining was performed using the Human Th17/Treg Phenotyping Kit (BD Bioscience) protocol as follows: 1) Clumps of cells and/or debris were removed by passing the cell suspension through a BD FalconTM 70-μm nylon cell strainer (Cat. No. 352235). 2) The cells were diluted to 10 million cells/ml. 3) 1 million cells were aliquoted per well and centrifuged at 300 x g for 5 minutes. Subsequently, the buffer was removed. 4) To fix the cells, the cell pellets were gently suspended in the residual volume of the staining buffer and 200 μl of freshly prepared cold 1 × BD PharmingenTM Mouse Foxp3 Fixation Buffer (provided, Cat. No. 560409) was added. The solution was mixed well and incubated for 30 minutes at 4°C in the dark. 5) The mixture was centrifuged at 300 x g for 5 minutes, and the fixative was removed. 6) To wash the cells, each pellet was suspended in 200 μl of freshly prepared pre-warmed (37°C) 1 × BD PharmingenTM Mouse Foxp3 Permeabilization Buffer (provided, Cat. No. 560409) and centrifuged 300 x g for 5 minutes. The permeabilization buffer was then removed. 7) To permeabilize the cells, the cell pellets were gently suspended in another 200 μl of freshly prepared, pre-warmed (37°C) 1 × BD PharmingenTM. 8) The cells were then centrifuged at 300 x g for 5 minutes and the Mouse Foxp3 Permeabilization Buffer was removed. The solution was incubated for 30 minutes at 37°C in the dark. 9) To wash cells, 200 μl of BD PharmingenTM stain buffer (FBS) was added to each tube and centrifuged at 300 x g for 5 minutes. The buffer was then removed and the process repeated. 10) 20 μl/test of the mouse Th17/Treg phenotyping cocktail or appropriate negative staining control was added. The mixture was incubated at RT for 30 minutes in the dark. Cells should be protected from light throughout staining and storage. 11) Wash step 9 was repeated twice. 12) The cell pellets were suspended in 200 μl of stain buffer, and flow cytometric analysis was performed. Staining was performed using 7-AAD (BD Bioscience) to check cell viability. 100 µl (1 x 105) of cell suspension was added with 3 µl 7-AAD and incubated at room temperature in the dark for 30 minutes. 1.5. Laboratory Investigations Complete blood count, plasma glucose level, and creatinine were assessed at the outpatient clinic. The glomerular filtration rate was calculated using the Cockroft-Gault formula. The neutrophil-to-lymphocyte ratio was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. 1.6. Statistical Analysis Continuous variables were expressed as mean ± standard deviation (SD) or median with interquartile ranges and were compared using the Student’s T-test or Mann-Whitney U test as appropriate. Categorical data were presented as frequency (percentages) and were compared using Fisher’s exact or chi-square test. All significance tests were two-tailed, rejecting the null hypothesis at p < 0.05. The correlation test was done using the Pearson or Spearman test, as appropriate. Confounding factors were identified by testing significant variables at univariate analysis with univariate logistic regression, followed by multicollinearity diagnostics. This intermediate step was introduced to select the variables to be included in a stepwise backward multivariate logistic regression model, providing odds ratios and 95% confidence intervals. All statistical analyses were performed using SPSS version 26.0 software (SPSS Inc., Chicago, IL, USA). RESULTS 2.1. Characteristics of the Study Population The subjects consisted of 40 participants from each group. The proportion of male subjects was lower than that of female subjects (25% vs. 75%, p < 0,001). The median age of subjects in the hypertension group was higher than that of the normotensive group (49 years old vs. 42 years old, p < 0,001). About half of the total sample were obese, and 72.5% of them were included in the group with essential hypertension. Only four subjects had controlled blood pressure with antihypertensive medication. The baseline characteristics can be seen in Table 1 . 2.2. Bivariate Analysis We conducted a bivariate analysis ( Table 2 ) to find the association between Treg and Th17 with hypertension. 2.2.1. Treg levels were lower in the hypertension group Treg levels were significantly lower in subjects with hypertension compared to those with normotension [23.25 (7.3–63.1)% vs. 65.05 (22.5–81.5)%, p < 0.001]. There was a strong negative correlation of Treg levels with systolic and diastolic blood pressure (r = -0.733 and r = -0.613, respectively; Figure 1 ). 2.2.2. Th17 and IL17A + FoxP3 + Treg levels were higher in the hypertension group Th17 was higher in the hypertension group compared to normotension [16.7 (0.2–36.2)% vs. 0.8 (0–13.8)%, p < 0.001]. There was a moderate correlation of Th17 cell levels with systolic and diastolic blood pressure, showing a positive pattern (r = 0.349 and r = 0.271, respectively; Figure 2 ). The levels of IL17A + FoxP3 + Treg were higher in the group with hypertension compared to the normotension group [41.6 (21.9–81.7)% vs. 20.7 (0.4–73.7)%, p < 0.001]. There was a moderate to strong correlation of IL17A + FoxP3 + Treg levels with both systolic and diastolic blood pressure, showing a positive pattern (r = 0.517 and r = 0.346, respectively; Figure 3 ). 2.2.3. The ratios of Th17/Treg and IL17A + FoxP3 + Treg/Treg were higher in the hypertension group We found a significant difference in the ratio of Th17/Treg between subjects with hypertension and those with normotension [0.726 (0.03–­4.91) vs. 0.012 (0.000­–0.563), p < 0.001]. There was a low to moderate correlation of the Th17/Treg ratio with both systolic and diastolic blood pressure, showing a positive pattern (r = 0.357 and r = 0.242, respectively; Figure 4 ). A significant difference in the ratio of IL17A + FoxP3 + Treg/Treg between subjects with hypertension and those with normotension was also obtained [2.11 (0.53–5.96) vs. 0.31 (0.01–3.27), p < 0.001]. There was a moderate to strong correlation of the IL17A + FoxP3 + Treg/Treg ratio with both systolic and diastolic blood pressure, showing a positive pattern (r = 0.607 and r = 0.386, respectively; Figure 5 ). 2.3. Multivariate Analysis After controlling other variables such as the ratio of Th17/Treg, abdominal circumference, sodium consumption, and eGFR in a multivariate analysis ( Table 3 ), we found that low Treg was associated with hypertension (OR = 0.81). DISCUSSION This study aimed to elaborate on the association between Treg and Th17 in human hypertension. We found a strong association between low Treg and hypertension. In contrast, we found a moderate association between high Th17 and hypertension. Furthermore, there was a weak to moderate association between the Th17/Treg ratio and hypertension. Interestingly, we also found a subpopulation of IL-17A + foxp3 + Treg cells, which had a moderate to strong association with hypertension. Treg plays an immunosuppressive role that has been proven to preserve blood pressure and prevent organ damage in hypertensive animal models. Translating the role of Treg in animal hypertension models, this study confirms that Treg is also strongly associated with hypertension in humans. 3 The results further align with studies conducted by Alexander et al. and Gackowska et al. regarding the association between Treg and hypertension in humans. 9 , 10 Despite the strong evidence of Treg’s role in animal studies conducted by Kvakan et al., where he demonstrated the ability of Treg injection to reduce blood pressure in hypertensive mice, we were not able to prove this causal relationship mechanism between Treg and hypertension in humans. 11 Hence, further investigation is warranted to prove the direct effect of Treg on human hypertension. Several studies have shown the importance of Th17 in hypertension. A study conducted by Madhur et al. revealed the relationship between Th17 and hypertension in mice. 8 Furthermore, a study by Orejudo et al. consistently displayed that Th17 cells produced an abundance of IL-17A in subjects with hypertensive nephropathy. 12 Our study was in agreement with these animal studies’ findings and revealed that higher Th17 was also associated with human hypertension. However, we noticed that the association between Th17 and hypertension was weaker compared to that between Treg and hypertension. Therefore, we postulate that Treg might have a greater role in human hypertension than Th17. Previous animal studies revealed the importance of Th17/Treg balance in the regulation of immune disorders, where Treg cells serve to suppress inflammatory processes while Th17 cells enhance immune activity. A study by Xie et al. showed that the ratio of Th17/Treg balance was important to atherosclerosis formation. 13 Another study by Anna et al. found that the ratio of Th17/Treg was significantly increased in patients with hypertension associated with hyperaldosteronism. 14 In agreement with these studies, we found that the Th17/Treg ratio had a weak association with human hypertension, which was possibly caused by the low Th17 population in this study. There were limited studies that investigated the role of IL-17A + foxp3 + Treg cell populations in hypertension. These cells had characteristics similar to Treg, with additional expression of ROR \(\:\gamma\:\) t transcription factors that could induce the pro-inflammatory cytokine IL-17A. These cells also have a role in the development of several disorders, including cancer, infectious diseases, and inflammatory diseases. The Treg cells malfunction and exhibit low suppressive action in psoriasis. Psoriasis patients were the first to have IL-17A-producing, Th17-like Treg cells found in human inflammatory diseases. In the human psoriatic skin, CD4 + Foxp3 + T cells display a memory phenotype by expressing CD45RO and demonstrating a high level of IL-17A, IL-2, and IFN-γ production. A significant portion of CD4 + Foxp3 + Treg cells releases pro-inflammatory cytokines, including IL-17A and IFN-γ, according to recent investigations and assessments of human tumor-infiltrating lymphocytes in melanoma, ovarian, breast, and colon malignancies. 15 , 16 A study by Voo et al. found that this cell was activated by several cytokines, such as IL-1 \(\:\beta\:\) and IL-6 in the periphery. 17 Beriou et al. reported that IL17A + FoxP3 + Treg cells retain suppressive function. However, during inflammatory conditions, these cells have the plasticity to produce IL-17A and might lose their suppressive ability. 18 Interestingly, in this study, we found a moderate to strong association between high IL17A + FoxP3 + Treg cells and hypertension. Therefore, we postulated in this study that the activation of Treg into IL17A + FoxP3 + Treg cells might be associated with blood pressure dysregulation. LIMITATION This study was designed to examine the association between Treg and Th17 cells with hypertension. Therefore, as a limitation of our study, we were not able to demonstrate the direct mechanism of these cells in hypertension. CONCLUSION Lower Treg and higher Th17 levels are associated with hypertension. This study also reported new evidence of IL-17A + foxp3 + Treg cell association in human hypertension. Further study is required to elaborate on the direct mechanism of these cells in human hypertension. Abbreviations Ang II Angiotensin II eGFR Estimated glomerular filtration rate FOX Forkhead box HBPM Home blood pressure monitoring IL Interleukin OR Odds ratio PBMC Peripheral blood mononuclear cell SD Standard deviation TGF Transforming growth factor Th17 T helper-17 Treg Regulatory T-cell Declarations Funding : This study was funded by a PUTI Universitas Indonesia grant. Conflict of Interest: None declared. DATA AVAILIBILITY Data is provided within the manuscript or supplementary information files. ETHICS APPROVAL AND CONSENT TO PARTICIPATE The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) and approved by the National Cardiovascular Centre of Harapan Kita Hospital and Universitas Indonesia Hospital Ethics Committee. Eligible participants provided written informed consent before participating in this study. CONSENT FOR PUBLICATION Not applicable COMPETING INTEREST The authors declare no competing interests. FUNDING This study was funded by a PUTI Universitas Indonesia grant. ACKNOWLEDGMENTS The authors acknowledge the tremendous help of the Indonesian Medical Education and Research Institute (IMERI), the National Cardiovascular Center Harapan Kita Laboratory, the National Cardiovascular Center Harapan Kita Research Unit, and the staff at the Universitas Indonesia Hospital Laboratory. CONFLICT OF INTEREST The authors declare no conflict of interests. AUTHOR CONTRIBUTIONS HSM proposed the study idea. HSM, SDP, BW, DZ, SNS, VR, BBS, NE, GRTR, and HZA planned the technique, contributed to the intellectual content, and critically assessed the manuscript. The manuscript was finalized by HSM, BW, and SDP. HSM, BW, and SDP carried out the statistical analysis. References Drummond GR, Vinh A, Guzik TJ, Sobey CG. Immune mechanisms of hypertension. Nat Rev Immunol . 2019;19(8):517–532. doi:10.1038/s41577-019-0160-5 Du Y-N, Tang X-F, Xu L, Gao PJ, Han WQ. Th17/Treg imbalance in hypertension. Cardiovasc Pharmacol Open Access . 2018;07(03):17–19. doi:10.4172/2329-6607.1000241 Viel EC, Lemarié CA, Benkirane K, Paradis P, Schiffrin EL. Immune regulation and vascular inflammation in genetic hypertension. Am J Physiol Heart Circ Physiol . 2010;298(3):938–945. doi:10.1152/ajpheart.00707.2009 Barhoumi T, Kasal DA, Li MW, et al. T Regulatory lymphocytes prevent angiotensin II-induced hypertension and vascular injury. Hypertension . 2011;57(3):469–476. doi:10.1161/HYPERTENSIONAHA.110.162941 Kasal DA, Barhoumi T, Li MW, et al. T regulatory lymphocytes prevent aldosterone-induced vascular injury. Hypertension . 2012;59(2):324–330. doi:10.1161/HYPERTENSIONAHA.111.181123 Mian MOR, Barhoumi T, Briet M, Paradis P, Schiffrin EL. Deficiency of T-regulatory cells exaggerates angiotensin II-induced microvascular injury by enhancing immune responses. J Hypertens . 2016;34(1):97–108. doi:10.1097/HJH.0000000000000761 De Ciuceis C, Rossini C, Airò P, et al. Relationship between different subpopulations of circulating CD4+ T-lymphocytes and microvascular structural alterations in humans. Am J Hypertens . 2017;30(1):51–60. doi:10.1093/ajh/hpw102 Madhur MS, Lob HE, McCann LA, et al. Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction. Hypertension . 2010;55(2):500–507. doi:10.1161/HYPERTENSIONAHA.109.145094 Alexander MR, Dale BL, Smart CD, et al. Immune Profiling Reveals Decreases in Circulating Regulatory and Exhausted T Cells in Human Hypertension. JACC Basic to Transl Sci . Published online 2023. doi:10.1016/j.jacbts.2022.09.007 Gackowska L, Michalkiewicz J, Helmin-Basa A, et al. Regulatory T-cell subset distribution in children with primary hypertension is associated with hypertension severity and hypertensive target organ damage. J Hypertens . 2020;38(4):692–700. doi:10.1097/HJH.0000000000002328 Kvakan H, Kleinewietfeld M, Qadri F, et al. Regulatory T cells ameliorate angiotensin II-induced cardiac damage. Circulation . 2009;119(22):2904–2912. doi:10.1161/CIRCULATIONAHA.108.832782 Orejudo M, Rodrigues-Diez RR, Rodrigues-Diez R, et al. Interleukin 17A participates in renal inflammation associated to experimental and human hypertension. Front Pharmacol . 2019;10(September):1–18. doi:10.3389/fphar.2019.01015 Xie J jiao, Wang J, Tang T ting, et al. The Th17/Treg functional imbalance during atherogenesis in ApoE-/- mice. Cytokine . 2010;49(2):185–193. doi:10.1016/j.cyto.2009.09.007 Imiela AM, Mikołajczyk TP, Siedliński M, et al. The Th17/Treg imbalance in patients with primary hyperaldosteronism and resistant hypertension. Polish Arch Intern Med . Published online 2021:1–10. doi:10.20452/pamw.16171 Du R, Zhao H, Yan F, Li H. IL-17 + Foxp3 + T cells: an intermediate differentiation stage between Th17 cells and regulatory T cells. J Leukoc Biol . 2014;96(1):39–48. doi:10.1189/jlb.1ru0114-010rr Jung MK, Kwak JE, Shin EC. 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Subject’s Characteristics Variables Total (N = 80) Hypertension status P value Normotension (40) Hypertension (40) Age (Years) 46 (24–60) 42.5 (24–57) 49 (30–60) <0.001* Sex, n (%) 0.439 Male 20 (25) 8 (20) 12 (30) Female 60 (75) 32 (80) 28 (70) Systolic BP (mmHg) 121.5 (91–197) 113 (91–128) 146 (120–197) <0.001* Diastolic BP (mmHg) 80 (65–118) 77 (65–82) 90 (74–118) <0.001* Abdominal waist (cm) 94.6 ± 10.5 90.2 ± 9.0 99.1 ± 10.0 <0.001* Intake Parameter Sodium (mg) 1535.7 (907.8–5679.1) 1425.6 (907.8–5072.2) 1675.4 (926.3–5679.1) 0.043* Calorie (kcal) 1731.1 ± 517.6 1633. 8 ± 423.1 1828.3 ± 586.7 0.093 Risk Factor, n (%) Smoker 1 (1.3) 0 (0) 1 (2.5) 1.00 Obesity 40 (50) 11 (27.5) 29 (72.5) <0.001* Treatment, n (%) ACEi/ARB 32 (40) 0 (0) 32 (80) CCB 10 (12.5) 0 (0) 10 (25) HCT 5 (6.3) 0 (0) 5 (12.5) Betablocker 17 (21.3) 0 (0) 17 (42.5) Laboratory Parameter Leukocyte 7774.0 ± 1776.3 7781.0 ± 1656.0 7766.9 ± 1910.2 0.972 Neutrophil to lymphocyte ratio (NLR) 1.895 (0.9–6.22) 1.84 (0.98–4.71) 1.65 (0.9–6.22) 0.345 Creatinine 0.8 (0.5–1.4) 0.7 (0.5–1.1) 0.8 (0.5–1.4) 0.034* eGFR 100 (51–129) 104 (59–129) 92.5 (51–119) 0.003* *Values are presented as mean standard deviation or median (minimum-maximum) as appropriate. Statistically significant if p-value < 0.05. Table 2. Bivariate Analysis Variable (Percentage) Total (N = 80) Hypertension (n = 40) Normotension (n = 40) P value a Treg Cell 41.5 (7.3–81.5) 23.25 (7.3–63.1) 65.05 (22.5–81.5) <0.001 Th17 Cell 2.1 (0–36.2) 16.7 (0.2–36.2) 0.8 (0–13.8) <0.001 IL17A + FoxP3 + Treg Cell 34.5 (0.4–81.7) 41.6 (21.9–81.7) 20.7 (0.4–73.7) < 0.001 Th17/Treg Ratio 0.036 (0.001–4.9100) 0.726 (0.03–4.91) 0.0115 (0.000–0.563) <0.001 FoxP3 + IL17A + Treg/ Treg Ratio 0.92 (0.01–5.96) 2.11 (0.53–5.96) 0.31 (0.01–3.27) < 0.001 *Descriptive data are presented as median (minimum-maximum) and are analyzed with Mann-Whitney test Table 3. Multivariate Analysis Variables β SE OR (95% CI) P value Regulatory T Cell (%) -0.216 0.062 0.81 (0.71–0.91) <0.001* Abdominal waist 0.248 0.079 1.282 (1.10–1.50) 0.002* Sodium 0.02 0.01 1.002 (1.00–1.04) 0.034* eGFR -0.115 0.047 0.89 (0.81–0.98) 0.014* T Helper 17 Cells/Regulatory T-cells Ratio -2.120 1.350 0.12 (0.01–1.69) 0.116 Additional Declarations No competing interests reported. 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Teguh","lastName":"Ryanto","suffix":""},{"id":503530231,"identity":"65bc9d59-8c01-4a2d-8739-99e4843979fd","order_by":8,"name":"Hilman Zulkifli Amin","email":"","orcid":"","institution":"National Cerebral and Cardiovascular Center","correspondingAuthor":false,"prefix":"","firstName":"Hilman","middleName":"Zulkifli","lastName":"Amin","suffix":""},{"id":503530235,"identity":"cb105272-da69-4402-b460-93f747c4cfec","order_by":9,"name":"Hary Sakti Muliawan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIie2RP0vDQBTA3xG5LJd2fUeK/QopgkWU+lUSAtelQ8cOQd8Ul+LsxzkpZKp0rWMoZHLIJBlUvKQuBS/g5nC/4Y7Hux/vzwE4HP+Q6HjFwH2CetWFjNpTgO5XkAvNnrZ/UgBjo5zkLMrUf6lkk6m7gTw8k17OzgFTgiaD0cCiXK3nl6EoFshDlZCO0gvAhNi6ACFtjWnFQ+AroyymhzrSCRkFAgIR2ZRdxWXzZRS5nZgq+r5V2Gefslccg9w0hqJT4rYxr7fKvvKug0clc3GcZZKLkjajAu2z7BR7bd7T4fhhYzb2MRsP/XlZvmU3t7aNGc7wJOTQ/Qj++vYHr+7LOhwOhwO+AY7bWPGCRoLcAAAAAElFTkSuQmCC","orcid":"","institution":"Universitas Indonesia","correspondingAuthor":true,"prefix":"","firstName":"Hary","middleName":"Sakti","lastName":"Muliawan","suffix":""}],"badges":[],"createdAt":"2025-05-10 15:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6635766/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6635766/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89672729,"identity":"77f99054-8fe5-4ab1-9a7a-25d8f6a5aa87","added_by":"auto","created_at":"2025-08-22 13:11:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Correlation between Treg and Blood Pressure\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6635766/v1/56dd740ef82051dc3e808216.png"},{"id":89673485,"identity":"85e2f2ac-9aa8-4932-ada1-1bf84d234e43","added_by":"auto","created_at":"2025-08-22 13:19:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Correlation between Th17 and Blood Pressure\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6635766/v1/f7f5f6fe3e231c784853c6f7.png"},{"id":89672732,"identity":"68409b8c-716b-4b05-8ebf-328eca0c18eb","added_by":"auto","created_at":"2025-08-22 13:11:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Correlation between IL17A\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eFoxP3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e Treg Cells and Blood Pressure\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6635766/v1/0d65668f007d3902ec08eeba.png"},{"id":89672734,"identity":"5c2ab1c1-7ec9-4ce3-b311-a139e6faa96b","added_by":"auto","created_at":"2025-08-22 13:11:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46186,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Correlation between the ratio of Th17/Treg Cell and Blood Pressure\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6635766/v1/617370c306624d6dd03507ef.png"},{"id":89672738,"identity":"ce45e008-7281-4fe9-9b2b-99123128e07e","added_by":"auto","created_at":"2025-08-22 13:11:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49508,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe Correlation between the ratio of IL17A\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eFoxP3\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eTreg/Treg Cell and Blood Pressure\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6635766/v1/141fc64677682c01c46e75d5.png"},{"id":89675360,"identity":"84648b5c-e555-419d-a0c7-b62f0c3e5fa8","added_by":"auto","created_at":"2025-08-22 13:35:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1355363,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6635766/v1/4b414d19-6cef-434e-a69b-a844b97b393e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Association Between Regulatory T-Cells and T Helper 17 Cells with Blood Pressure in Hypertension","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe discovery of autoantibodies in artery samples from “hypertensive cadavers” more than 50 years ago raised the possibility of a relationship between the immune system and hypertension.\u003csup\u003e1\u003c/sup\u003e Since then, various studies on the role of the immune system in hypertension have emerged. Animal studies revealed that an imbalance of the T helper-17 (Th17) cell to Regulatory T-cell (Treg) ratio plays a critical role in hypertension.\u003csup\u003e2\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTreg was first discovered as a novel regulator of hypertension through a study conducted by Dr. Schiffrin’s team. Using consomic-modified Brown Norway rats of normotensive strain chromosome 2\u0026nbsp;with the Dahl salt-sensitive genome, which overexpresses Treg cells, they found lower blood pressure and vascular hypertrophy compared to Dahl salt-sensitive rats. The consomic rats have also increased production of the anti-inflammatory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-\u003cimg width=\"10\" height=\"37\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAAoAAAAlCAMAAABMFohsAAAAAXNSR0IArs4c6QAAAF1QTFRFAAAAAAAAAAA6AABmADpmADqQAGa2OgAAOgA6OpDbZgAAZgA6ZpBmZpDbZrbbZrb/kDoAkNv/tmYAtmY6ttv/tv//25A625Bm27Zm2////7Zm/9uQ/9u2//+2///bGQ1xbAAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAADsQAAA7EAZUrDhsAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAYUlEQVQoU91OSRKAMAhr616X4la1av//TCE9ePZqZshkIAGU+oit0sYh47OFa2V1W+4ESPBUypw5zrk0I7lgeoTOelSXHUTC6osjWZlERmLedSNb245vvdb0qMdugMT1TzzfkwQ9d0eN7AAAAABJRU5ErkJggg==\" alt=\"image\"\u003e.\u003csup\u003e3\u003c/sup\u003e Another study conducted by Barhoumi demonstrated the ability of Treg to inhibit angiotensin II-mediated vascular damage. In contrast, angiotensin II-induced a significant reduction of Treg in the renal cortex.\u003csup\u003e4\u003c/sup\u003e In concordance with Barhoumi’s findings, Kasat et al. also showed that Treg inhibits aldosterone-mediated vascular injury through the interaction between innate and adaptive immunity.\u003csup\u003e5\u003c/sup\u003e Moreover, Mian et al. showed that Treg insufficiency amplified angiotensin II-induced microvascular damage.\u003csup\u003e6\u003c/sup\u003e Furthermore, a human study by Ciuceis et al. revealed that several lymphocytes, including Treg, were associated with microvascular remodeling.\u003csup\u003e7\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;A recent study also showed the importance of Th17 cells in hypertension. A study conducted by Madhur et al.\u0026nbsp;demonstrated a direct correlation between Th17 cells and angiotensin II (Ang II)-induced hypertension where they found preservation of vascular structure, decreased superoxide production, and low aortic T-cell infiltration in IL-17 null mice compared with wild-type.\u003csup\u003e8\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBased on the emerging evidence of the Th17/Treg cell role in animal hypertension studies, this study investigated the association of those cells with human hypertension.\u0026nbsp;\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e1.1. Study Design and Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is a case-control study conducted from August 2022 to December 2022. All patients admitted to the outpatient clinic in the National Cardiovascular Center of Harapan Kita and Universitas Indonesia Hospital were screened for eligibility. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval by the institution\u0026rsquo;s human research committee and board committee ethics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2. \u003c/strong\u003e \u003cstrong\u003eSubject Recruitment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe included all patients aged 18 years or older who were admitted to the outpatient clinic with essential hypertension or normotension. We excluded patients with diabetes mellitus, coronary heart disease, stroke, peripheral artery disease, heart failure, chronic kidney disease, infection, cancer, autoimmune disease, and statin therapy. Informed consent was obtained from each included patient.\u003c/p\u003e\n\u003cp\u003eEach patient who was admitted to the outpatient clinic was screened with a history, physical examination, blood pressure measurement, waist circumference measurement, and body mass index assessment. Blood pressure was measured with a calibrated digital OMRON\u003csup\u003eTM\u003c/sup\u003e HEM 8712 using JNC 8 protocols. Body weight was measured with a calibrated Camry weighing scale using the standardized protocol. Height was measured with a standardized Microtoise. Waist circumference was measured with a measuring tape measure using the World Health Organization protocol. Selected subjects then underwent further examination, including a dietary habit intake interview and a blood examination. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.3. Hypertension and Dietary Intake Determination \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOffice blood pressure measurement was taken during the first meeting. Additionally, for each participant, home blood pressure measurements (HBPM) were conducted over a period of five days. Participants measured their blood pressure twice a day: in the morning when they woke up and at night when they were going to sleep. Blood pressure was measured with a calibrated digital OMRON\u003csup\u003eTM\u003c/sup\u003e HEM 8712 using JNC 8 protocols.\u003c/p\u003e\n\u003cp\u003eThe food frequency and 24-hour recall questionnaires were utilized for calculating dietary intake. The 24-hour recall was conducted by two trained enumerators who assisted interviewees with scripts, visuals, and other tools, including measuring cups and food models, while conducting interviews in Indonesian. The food preparation process, brand names (if applicable), and supplements utilized were all noted thoroughly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.4. \u003c/strong\u003e \u003cstrong\u003eBlood Sample Collection and Immunological Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe blood suspension was collected in a vacutainer tube coated with EDTA. The blood was then processed for peripheral blood mononuclear cell (PBMC) isolation by density gradient centrifugation using Lymphoprep\u0026trade; (StemCell Technologies). Blood was mixed with Lymphoprep\u0026trade; in a 1:1 volume. The mixture was then centrifuged at 800 x g for 20 minutes at 20\u0026deg;C. The part of the buffy coat that was under the plasma was taken and resuspended with a complete PBMC medium for examination using flow cytometry. Flow cytometry examination was done using the FACS ARIA III (BD Bioscience) tool. For examining IL-17A and Foxp3 markers, staining was performed using the Human Th17/Treg Phenotyping Kit (BD Bioscience) protocol as follows: \u003c/p\u003e\n\u003cp\u003e1) Clumps of cells and/or debris were removed by passing the cell suspension through a BD FalconTM 70-\u0026mu;m nylon cell strainer (Cat. No. 352235).\u003c/p\u003e\n\u003cp\u003e2) The cells were diluted to 10 million cells/ml.\u003c/p\u003e\n\u003cp\u003e3) 1 million cells were aliquoted per well and centrifuged at 300 x g for 5 minutes. Subsequently, the buffer was removed.\u003c/p\u003e\n\u003cp\u003e4) To fix the cells, the cell pellets were gently suspended in the residual volume of the staining buffer and 200 \u0026mu;l of freshly prepared cold 1 \u0026times; BD PharmingenTM Mouse Foxp3 Fixation Buffer (provided, Cat. No. 560409) was added. The solution was mixed well and incubated for 30 minutes at 4\u0026deg;C in the dark.\u003c/p\u003e\n\u003cp\u003e5) The mixture was centrifuged at 300 x g for 5 minutes, and the fixative was removed.\u003c/p\u003e\n\u003cp\u003e6) To wash the cells, each pellet was suspended in 200 \u0026mu;l of freshly prepared pre-warmed (37\u0026deg;C) 1 \u0026times; BD PharmingenTM Mouse Foxp3 Permeabilization Buffer (provided, Cat. No. 560409) and centrifuged 300 x g for 5 minutes. The permeabilization buffer was then removed.\u003c/p\u003e\n\u003cp\u003e7) To permeabilize the cells, the cell pellets were gently suspended in another 200 \u0026mu;l of freshly prepared, pre-warmed (37\u0026deg;C) 1 \u0026times; BD PharmingenTM.\u003c/p\u003e\n\u003cp\u003e8) The cells were then centrifuged at 300 x g for 5 minutes and the Mouse Foxp3 Permeabilization Buffer was removed. The solution was incubated for 30 minutes at 37\u0026deg;C in the dark.\u003c/p\u003e\n\u003cp\u003e9) To wash cells, 200 \u0026mu;l of BD PharmingenTM stain buffer (FBS) was added to each tube and centrifuged at 300 x g for 5 minutes. The buffer was then removed and the process repeated.\u003c/p\u003e\n\u003cp\u003e10) 20 \u0026mu;l/test of the mouse Th17/Treg phenotyping cocktail or appropriate negative staining control was added. The mixture was incubated at RT for 30 minutes in the dark. Cells should be protected from light throughout staining and storage.\u003c/p\u003e\n\u003cp\u003e11) Wash step 9 was repeated twice. \u003c/p\u003e\n\u003cp\u003e12) The cell pellets were suspended in 200 \u0026mu;l of stain buffer, and flow cytometric analysis was performed.\u003c/p\u003e\n\u003cp\u003eStaining was performed using 7-AAD (BD Bioscience) to check cell viability. 100 \u0026micro;l (1 x 105) of cell suspension was added with 3 \u0026micro;l 7-AAD and incubated at room temperature in the dark for 30 minutes. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.5. Laboratory Investigations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplete blood count, plasma glucose level, and creatinine were assessed at the outpatient clinic. The glomerular filtration rate was calculated using the Cockroft-Gault formula. The neutrophil-to-lymphocyte ratio was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.6. \u003c/strong\u003e\u003cstrong\u003e Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContinuous variables were expressed as mean \u0026plusmn; standard deviation (SD) or median with interquartile ranges and were compared using the Student\u0026rsquo;s T-test or Mann-Whitney U test as appropriate. Categorical data were presented as frequency (percentages) and were compared using Fisher\u0026rsquo;s exact or chi-square test. All significance tests were two-tailed, rejecting the null hypothesis at p \u0026lt; 0.05. The correlation test was done using the Pearson or Spearman test, as appropriate.\u003c/p\u003e\n\u003cp\u003eConfounding factors were identified by testing significant variables at univariate analysis with univariate logistic regression, followed by multicollinearity diagnostics. This intermediate step was introduced to select the variables to be included in a stepwise backward multivariate logistic regression model, providing odds ratios and 95% confidence intervals. All statistical analyses were performed using SPSS version 26.0 software (SPSS Inc., Chicago, IL, USA).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e2.1. Characteristics of the Study Population\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe subjects consisted of 40 participants from each group. The proportion of male subjects was lower than that of female subjects (25% vs. 75%, p \u0026lt; 0,001). The median age of subjects in the hypertension group was higher than that of the normotensive group (49 years old vs. 42 years old, p \u0026lt; 0,001). About half of the total sample were obese, and 72.5% of them were included in the group with essential hypertension. Only four subjects had controlled blood pressure with antihypertensive medication. The baseline characteristics can be seen in \u003cstrong\u003eTable 1\u003c/strong\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Bivariate Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe conducted a bivariate analysis (\u003cstrong\u003eTable 2\u003c/strong\u003e) to find the association between Treg and Th17 with hypertension. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.1. Treg levels were lower in the hypertension group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTreg levels were significantly lower in subjects with hypertension compared to those with normotension [23.25 (7.3–63.1)% vs. 65.05 (22.5–81.5)%, p \u0026lt; 0.001]. There was a strong negative correlation of Treg levels with systolic and diastolic blood pressure (r = -0.733 and r = -0.613, respectively; \u003cstrong\u003eFigure 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.2. \u003c/strong\u003e \u003cstrong\u003eTh17 and \u003c/strong\u003e\u003cstrong\u003eIL17A\u003csup\u003e+\u003c/sup\u003eFoxP3\u003csup\u003e+\u003c/sup\u003eTreg levels were higher in the hypertension group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTh17 was higher in the hypertension group compared to normotension [16.7 (0.2–36.2)% vs. 0.8 (0–13.8)%, p \u0026lt; 0.001]. There was a moderate correlation of Th17 cell levels with systolic and diastolic blood pressure, showing a positive pattern (r = 0.349 and r = 0.271, respectively; \u003cstrong\u003eFigure 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe levels of IL17A\u003csup\u003e+\u003c/sup\u003eFoxP3\u003csup\u003e+\u003c/sup\u003eTreg were higher in the group with hypertension compared to the normotension group [41.6 (21.9–81.7)% vs. 20.7 (0.4–73.7)%, p \u0026lt; 0.001]. There was a moderate to strong correlation of IL17A\u003csup\u003e+\u003c/sup\u003eFoxP3\u003csup\u003e+\u003c/sup\u003eTreg levels with both systolic and diastolic blood pressure, showing a positive pattern (r = 0.517 and r = 0.346, respectively; \u003cstrong\u003eFigure 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2.3. The ratios of Th17/Treg and IL17A\u003csup\u003e+\u003c/sup\u003eFoxP3\u003csup\u003e+\u003c/sup\u003eTreg/Treg were higher in the hypertension group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe found a significant difference in the ratio of Th17/Treg between subjects with hypertension and those with normotension [0.726 (0.03–­4.91) vs. 0.012 (0.000­–0.563), p \u0026lt; 0.001]. There was a low to moderate correlation of the Th17/Treg ratio with both systolic and diastolic blood pressure, showing a positive pattern (r = 0.357 and r = 0.242, respectively; \u003cstrong\u003eFigure 4\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eA significant difference in the ratio of IL17A\u003csup\u003e+\u003c/sup\u003eFoxP3\u003csup\u003e+\u003c/sup\u003eTreg/Treg between subjects with hypertension and those with normotension was also obtained [2.11 (0.53–5.96) vs. 0.31 (0.01–3.27), p \u0026lt; 0.001]. There was a moderate to strong correlation of the IL17A\u003csup\u003e+\u003c/sup\u003eFoxP3\u003csup\u003e+\u003c/sup\u003eTreg/Treg ratio with both systolic and diastolic blood pressure, showing a positive pattern (r = 0.607 and r = 0.386, respectively; \u003cstrong\u003eFigure 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Multivariate Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter controlling other variables such as the ratio of Th17/Treg, abdominal circumference, sodium consumption, and eGFR in a multivariate analysis (\u003cstrong\u003eTable 3\u003c/strong\u003e), we found that low Treg was associated with hypertension (OR = 0.81).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study aimed to elaborate on the association between Treg and Th17 in human hypertension. We found a strong association between low Treg and hypertension. In contrast, we found a moderate association between high Th17 and hypertension. Furthermore, there was a weak to moderate association between the Th17/Treg ratio and hypertension. Interestingly, we also found a subpopulation of IL-17A\u003csup\u003e+\u003c/sup\u003efoxp3\u003csup\u003e+\u003c/sup\u003eTreg cells, which had a moderate to strong association with hypertension.\u003c/p\u003e\u003cp\u003eTreg plays an immunosuppressive role that has been proven to preserve blood pressure and prevent organ damage in hypertensive animal models. Translating the role of Treg in animal hypertension models, this study confirms that Treg is also strongly associated with hypertension in humans.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e The results further align with studies conducted by Alexander et al. and Gackowska et al. regarding the association between Treg and hypertension in humans.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Despite the strong evidence of Treg\u0026rsquo;s role in animal studies conducted by Kvakan et al., where he demonstrated the ability of Treg injection to reduce blood pressure in hypertensive mice, we were not able to prove this causal relationship mechanism between Treg and hypertension in humans.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Hence, further investigation is warranted to prove the direct effect of Treg on human hypertension.\u003c/p\u003e\u003cp\u003eSeveral studies have shown the importance of Th17 in hypertension. A study conducted by Madhur et al. revealed the relationship between Th17 and hypertension in mice.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Furthermore, a study by Orejudo et al. consistently displayed that Th17 cells produced an abundance of IL-17A in subjects with hypertensive nephropathy.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Our study was in agreement with these animal studies\u0026rsquo; findings and revealed that higher Th17 was also associated with human hypertension. However, we noticed that the association between Th17 and hypertension was weaker compared to that between Treg and hypertension. Therefore, we postulate that Treg might have a greater role in human hypertension than Th17.\u003c/p\u003e\u003cp\u003ePrevious animal studies revealed the importance of Th17/Treg balance in the regulation of immune disorders, where Treg cells serve to suppress inflammatory processes while Th17 cells enhance immune activity. A study by Xie et al. showed that the ratio of Th17/Treg balance was important to atherosclerosis formation.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Another study by Anna et al. found that the ratio of Th17/Treg was significantly increased in patients with hypertension associated with hyperaldosteronism.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e In agreement with these studies, we found that the Th17/Treg ratio had a weak association with human hypertension, which was possibly caused by the low Th17 population in this study.\u003c/p\u003e\u003cp\u003eThere were limited studies that investigated the role of IL-17A\u003csup\u003e+\u003c/sup\u003efoxp3\u003csup\u003e+\u003c/sup\u003e Treg cell populations in hypertension. These cells had characteristics similar to Treg, with additional expression of ROR\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\gamma\\:\\)\u003c/span\u003e\u003c/span\u003et transcription factors that could induce the pro-inflammatory cytokine IL-17A. These cells also have a role in the development of several disorders, including cancer, infectious diseases, and inflammatory diseases. The Treg cells malfunction and exhibit low suppressive action in psoriasis. Psoriasis patients were the first to have IL-17A-producing, Th17-like Treg cells found in human inflammatory diseases. In the human psoriatic skin, CD4\u0026thinsp;+\u0026thinsp;Foxp3\u0026thinsp;+\u0026thinsp;T cells display a memory phenotype by expressing CD45RO and demonstrating a high level of IL-17A, IL-2, and IFN-γ production. A significant portion of CD4\u0026thinsp;+\u0026thinsp;Foxp3\u0026thinsp;+\u0026thinsp;Treg cells releases pro-inflammatory cytokines, including IL-17A and IFN-γ, according to recent investigations and assessments of human tumor-infiltrating lymphocytes in melanoma, ovarian, breast, and colon malignancies.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e A study by Voo et al. found that this cell was activated by several cytokines, such as IL-1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e and IL-6 in the periphery.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Beriou et al. reported that IL17A\u0026thinsp;+\u0026thinsp;FoxP3\u0026thinsp;+\u0026thinsp;Treg cells retain suppressive function. However, during inflammatory conditions, these cells have the plasticity to produce IL-17A and might lose their suppressive ability.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Interestingly, in this study, we found a moderate to strong association between high IL17A\u0026thinsp;+\u0026thinsp;FoxP3\u0026thinsp;+\u0026thinsp;Treg cells and hypertension. Therefore, we postulated in this study that the activation of Treg into IL17A\u0026thinsp;+\u0026thinsp;FoxP3\u0026thinsp;+\u0026thinsp;Treg cells might be associated with blood pressure dysregulation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLIMITATION\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis study was designed to examine the association between Treg and Th17 cells with hypertension. Therefore, as a limitation of our study, we were not able to demonstrate the direct mechanism of these cells in hypertension.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eLower Treg and higher Th17 levels are associated with hypertension. This study also reported new evidence of IL-17A\u0026thinsp;+\u0026thinsp;foxp3\u0026thinsp;+\u0026thinsp;Treg cell association in human hypertension. Further study is required to elaborate on the direct mechanism of these cells in human hypertension.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAng II\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAngiotensin II\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eeGFR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEstimated glomerular filtration rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFOX\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eForkhead box\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHBPM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHome blood pressure monitoring\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterleukin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eOR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eOdds ratio\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBMC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePeripheral blood mononuclear cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStandard deviation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTGF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransforming growth factor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTh17\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eT helper-17\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTreg\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRegulatory T-cell\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This study was funded by a PUTI Universitas Indonesia grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eNone declared.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILIBILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Data is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) and approved by the National Cardiovascular Centre of Harapan Kita Hospital and Universitas Indonesia Hospital Ethics Committee. Eligible participants provided written informed consent before participating in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONSENT FOR PUBLICATION\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by a PUTI Universitas Indonesia grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the tremendous help of the Indonesian Medical Education and Research Institute (IMERI), the National Cardiovascular Center Harapan Kita Laboratory, the National Cardiovascular Center Harapan Kita Research Unit, and the staff at the Universitas Indonesia Hospital Laboratory.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; HSM proposed the study idea. HSM, SDP, BW, DZ, SNS, VR, BBS, NE, GRTR, and HZA planned the technique, contributed to the intellectual content, and critically assessed the manuscript. The manuscript was finalized by HSM, BW, and SDP. HSM, BW, and SDP carried out the statistical analysis.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDrummond GR, Vinh A, Guzik TJ, Sobey CG. Immune mechanisms of hypertension. \u003cem\u003eNat Rev Immunol\u003c/em\u003e. 2019;19(8):517\u0026ndash;532. doi:10.1038/s41577-019-0160-5\u003c/li\u003e\n \u003cli\u003eDu Y-N, Tang X-F, Xu L, Gao PJ, Han WQ. Th17/Treg imbalance in hypertension. \u003cem\u003eCardiovasc Pharmacol Open Access\u003c/em\u003e. 2018;07(03):17\u0026ndash;19. doi:10.4172/2329-6607.1000241\u003c/li\u003e\n \u003cli\u003eViel EC, Lemari\u0026eacute; CA, Benkirane K, Paradis P, Schiffrin EL. Immune regulation and vascular inflammation in genetic hypertension. \u003cem\u003eAm J Physiol Heart Circ Physiol\u003c/em\u003e. 2010;298(3):938\u0026ndash;945. doi:10.1152/ajpheart.00707.2009\u003c/li\u003e\n \u003cli\u003eBarhoumi T, Kasal DA, Li MW, et al. T Regulatory lymphocytes prevent angiotensin II-induced hypertension and vascular injury. \u003cem\u003eHypertension\u003c/em\u003e. 2011;57(3):469\u0026ndash;476. doi:10.1161/HYPERTENSIONAHA.110.162941\u003c/li\u003e\n \u003cli\u003eKasal DA, Barhoumi T, Li MW, et al. T regulatory lymphocytes prevent aldosterone-induced vascular injury. \u003cem\u003eHypertension\u003c/em\u003e. 2012;59(2):324\u0026ndash;330. doi:10.1161/HYPERTENSIONAHA.111.181123\u003c/li\u003e\n \u003cli\u003eMian MOR, Barhoumi T, Briet M, Paradis P, Schiffrin EL. Deficiency of T-regulatory cells exaggerates angiotensin II-induced microvascular injury by enhancing immune responses. \u003cem\u003eJ Hypertens\u003c/em\u003e. 2016;34(1):97\u0026ndash;108. doi:10.1097/HJH.0000000000000761\u003c/li\u003e\n \u003cli\u003eDe Ciuceis C, Rossini C, Air\u0026ograve; P, et al. Relationship between different subpopulations of circulating CD4+ T-lymphocytes and microvascular structural alterations in humans. \u003cem\u003eAm J Hypertens\u003c/em\u003e. 2017;30(1):51\u0026ndash;60. doi:10.1093/ajh/hpw102\u003c/li\u003e\n \u003cli\u003eMadhur MS, Lob HE, McCann LA, et al. Interleukin 17 promotes angiotensin II-induced hypertension and vascular dysfunction. \u003cem\u003eHypertension\u003c/em\u003e. 2010;55(2):500\u0026ndash;507. doi:10.1161/HYPERTENSIONAHA.109.145094\u003c/li\u003e\n \u003cli\u003eAlexander MR, Dale BL, Smart CD, et al. Immune Profiling Reveals Decreases in Circulating Regulatory and Exhausted T Cells in Human Hypertension. \u003cem\u003eJACC Basic to Transl Sci\u003c/em\u003e. Published online 2023. doi:10.1016/j.jacbts.2022.09.007\u003c/li\u003e\n \u003cli\u003eGackowska L, Michalkiewicz J, Helmin-Basa A, et al. Regulatory T-cell subset distribution in children with primary hypertension is associated with hypertension severity and hypertensive target organ damage. \u003cem\u003eJ Hypertens\u003c/em\u003e. 2020;38(4):692\u0026ndash;700. doi:10.1097/HJH.0000000000002328\u003c/li\u003e\n \u003cli\u003eKvakan H, Kleinewietfeld M, Qadri F, et al. Regulatory T cells ameliorate angiotensin II-induced cardiac damage. \u003cem\u003eCirculation\u003c/em\u003e. 2009;119(22):2904\u0026ndash;2912. doi:10.1161/CIRCULATIONAHA.108.832782\u003c/li\u003e\n \u003cli\u003eOrejudo M, Rodrigues-Diez RR, Rodrigues-Diez R, et al. Interleukin 17A participates in renal inflammation associated to experimental and human hypertension. \u003cem\u003eFront Pharmacol\u003c/em\u003e. 2019;10(September):1\u0026ndash;18. doi:10.3389/fphar.2019.01015\u003c/li\u003e\n \u003cli\u003eXie J jiao, Wang J, Tang T ting, et al. The Th17/Treg functional imbalance during atherogenesis in ApoE-/- mice. \u003cem\u003eCytokine\u003c/em\u003e. 2010;49(2):185\u0026ndash;193. doi:10.1016/j.cyto.2009.09.007\u003c/li\u003e\n \u003cli\u003eImiela AM, Mikołajczyk TP, Siedliński M, et al. The Th17/Treg imbalance in patients with primary hyperaldosteronism and resistant hypertension. \u003cem\u003ePolish Arch Intern Med\u003c/em\u003e. Published online 2021:1\u0026ndash;10. doi:10.20452/pamw.16171\u003c/li\u003e\n \u003cli\u003eDu R, Zhao H, Yan F, Li H. IL-17 + Foxp3 + T cells: an intermediate differentiation stage between Th17 cells and regulatory T cells. \u003cem\u003eJ Leukoc Biol\u003c/em\u003e. 2014;96(1):39\u0026ndash;48. doi:10.1189/jlb.1ru0114-010rr\u003c/li\u003e\n \u003cli\u003eJung MK, Kwak JE, Shin EC. IL-17A-producing foxp3+ regulatory T cells and human diseases. \u003cem\u003eImmune Netw\u003c/em\u003e. 2017;17(5):276\u0026ndash;286. doi:10.4110/in.2017.17.5.276\u003c/li\u003e\n \u003cli\u003eVoo KS, Wang Y-H, Santori FR, et al. Identification of IL-17-producing FOXP3+ regulatory T cells in humans. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e. 2009;106(12):4793\u0026ndash;4798. doi:10.1073/pnas.0900408106\u003c/li\u003e\n \u003cli\u003eBeriou G, Costantino CM, Ashley CW, et al. IL-17-producing human peripheral regulatory T cells retain suppressive function. \u003cem\u003eBlood\u003c/em\u003e. 2009;113(18):4240\u0026ndash;4249. doi:10.1182/blood-2008-10-183251\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Subject\u0026rsquo;s Characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"608\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal (N = 80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 41.7896%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHypertension status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormotension (40)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHypertension (40)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eAge (Years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e46 (24\u0026ndash;60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e42.5 (24\u0026ndash;57)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e49 (30\u0026ndash;60)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eSex, n (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e0.439\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e20 (25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e8 (20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e12 (30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e60 (75)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e32 (80)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e28 (70)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eSystolic BP (mmHg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e121.5 (91\u0026ndash;197)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e113 (91\u0026ndash;128)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e146 (120\u0026ndash;197)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eDiastolic BP (mmHg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e80 (65\u0026ndash;118)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e77 (65\u0026ndash;82)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e90 (74\u0026ndash;118)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eAbdominal waist (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e94.6 \u0026plusmn; 10.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e90.2 \u0026plusmn; 9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e99.1 \u0026plusmn; 10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIntake Parameter\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eSodium (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e1535.7 (907.8\u0026ndash;5679.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e1425.6 (907.8\u0026ndash;5072.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e1675.4 (926.3\u0026ndash;5679.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e0.043*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eCalorie (kcal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e1731.1 \u0026plusmn; 517.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e1633. 8 \u0026plusmn; 423.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e1828.3 \u0026plusmn; 586.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e0.093\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRisk Factor, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eSmoker\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e1 (1.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e1 (2.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eObesity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e40 (50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e11 (27.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e29 (72.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment, n (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eACEi/ARB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e32 (40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e32 (80)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eCCB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e10 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e10 (25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eHCT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e5 (6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e5 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eBetablocker\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e17 (21.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e17 (42.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLaboratory Parameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eLeukocyte\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e7774.0 \u0026plusmn; 1776.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e7781.0 \u0026plusmn; 1656.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e7766.9 \u0026plusmn; 1910.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e0.972\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eNeutrophil to lymphocyte ratio (NLR)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e1.895 (0.9\u0026ndash;6.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e1.84 (0.98\u0026ndash;4.71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e1.65 (0.9\u0026ndash;6.22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e0.345\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eCreatinine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e0.8 (0.5\u0026ndash;1.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e0.7 (0.5\u0026ndash;1.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e0.8 (0.5\u0026ndash;1.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e0.034*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 29.6624%;\"\u003e\n \u003cp\u003eeGFR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 18.0269%;\"\u003e\n \u003cp\u003e100 (51\u0026ndash;129)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.9767%;\"\u003e\n \u003cp\u003e104 (59\u0026ndash;129)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 20.8128%;\"\u003e\n \u003cp\u003e92.5 (51\u0026ndash;119)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 8.8496%;\"\u003e\n \u003cp\u003e0.003*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Values are presented as mean\u0026nbsp;\u003cimg width=\"11\" height=\"36\" src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAAsAAAAkBAMAAACteN32AAAAAXNSR0IArs4c6QAAABhQTFRFAAAAAAAAAAA6ZgA6kDoAkNv///+2///bgbS1+AAAAAF0Uk5TAEDm2GYAAAAJcEhZcwAADsQAAA7EAZUrDhsAAAAZdEVYdFNvZnR3YXJlAE1pY3Jvc29mdCBPZmZpY2V/7TVxAAAAI0lEQVQYV2NgwAuSA8DSWKgSQUFBIZAsbiVQk+Eq8VtFf1kAmh8HqxJscoAAAAAASUVORK5CYII=\" alt=\"image\"\u003e\u0026nbsp;standard deviation or median (minimum-maximum) as appropriate. Statistically significant if p-value \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Bivariate Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"643\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 179px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable (Percentage)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal (N = 80)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHypertension\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 40)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 147px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNormotension\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(n = 40)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreg Cell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e41.5 (7.3\u0026ndash;81.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e23.25 (7.3\u0026ndash;63.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e65.05 (22.5\u0026ndash;81.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTh17 Cell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e2.1 (0\u0026ndash;36.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e16.7 (0.2\u0026ndash;36.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e0.8 (0\u0026ndash;13.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL17A\u003csup\u003e+\u003c/sup\u003eFoxP3\u003csup\u003e+\u003c/sup\u003e Treg Cell\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e34.5 (0.4\u0026ndash;81.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e41.6 (21.9\u0026ndash;81.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e20.7 (0.4\u0026ndash;73.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTh17/Treg Ratio\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.036 (0.001\u0026ndash;4.9100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e0.726 (0.03\u0026ndash;4.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e0.0115 (0.000\u0026ndash;0.563)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt;0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFoxP3\u003csup\u003e+\u003c/sup\u003eIL17A\u003csup\u003e+\u003c/sup\u003eTreg/ Treg Ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 142px;\"\u003e\n \u003cp\u003e0.92 (0.01\u0026ndash;5.96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 102px;\"\u003e\n \u003cp\u003e2.11 (0.53\u0026ndash;5.96)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 147px;\"\u003e\n \u003cp\u003e0.31 (0.01\u0026ndash;3.27)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 73px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Descriptive data are presented as median (minimum-maximum) and are analyzed with Mann-Whitney test\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Multivariate Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"576\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariables\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026beta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSE\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 178px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOR (95% CI)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRegulatory T Cell (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-0.216\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.062\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 178px;\"\u003e\n \u003cp\u003e0.81 (0.71\u0026ndash;0.91)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u0026lt;0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAbdominal waist\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0.248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.079\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 178px;\"\u003e\n \u003cp\u003e1.282 (1.10\u0026ndash;1.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.002*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSodium\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 178px;\"\u003e\n \u003cp\u003e1.002 (1.00\u0026ndash;1.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.034*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eeGFR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-0.115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e0.047\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 178px;\"\u003e\n \u003cp\u003e0.89 (0.81\u0026ndash;0.98)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0.014*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 207px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eT Helper 17 Cells/Regulatory T-cells Ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 68px;\"\u003e\n \u003cp\u003e-2.120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003e1.350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 178px;\"\u003e\n \u003cp\u003e0.12 (0.01\u0026ndash;1.69)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e\u0026nbsp;0.116\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Regulatory T-cells, T helper-17 cells, hypertension, immune system","lastPublishedDoi":"10.21203/rs.3.rs-6635766/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6635766/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The immune system is currently been postulated to play a role in hypertension. The imbalance of Regulatory T-cells and T helper-17 cells has been found to be the cause of hypertension in animal studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod and Results: \u003c/strong\u003eThis case-control study was conducted at the National Cardiovascular Center of Harapan Kita and Universitas Indonesia Hospital from August to December 2022. Subjects with essential hypertension and normotension who visited the outpatient clinic were consecutively included. A flow cytometry examination was performed to quantify Regulatory T-cell and T helper-17 cell levels in the blood. The study consisted of 40 subjects from each group. The results showed that Regulatory T-cells were lower in the hypertension group compared to the normotension group (p \u0026lt; 0.001). We observed a strong correlation between Regulatory T-cells and both systolic blood pressure (r = -0.733, p \u0026lt; 0.001) and diastolic blood pressure (r = -0.613, p \u0026lt; 0.001). T helper-17 cells were also higher in the hypertension group (p \u0026lt; 0.001). Furthermore, we identified a moderate correlation between T helper-17 cells and systolic blood pressure (r = 0.357, p \u0026lt; 0.001) and diastolic blood pressure (r = 0.242 and p = 0.015). The ratio of T helper-17 cells to Regulatory T-cells was significantly higher (p \u0026lt; 0.001) in subjects with hypertension.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eWe revealed an association between Regulatory T-cells and T helper-17 cells in human hypertension. We further concluded that human hypertension is characterized by low Regulatory T-cells and high T helper-17 cells.\u003c/p\u003e","manuscriptTitle":"The Association Between Regulatory T-Cells and T Helper 17 Cells with Blood Pressure in Hypertension","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 13:11:22","doi":"10.21203/rs.3.rs-6635766/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-09-04T06:27:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-03T16:01:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299157789598309251322258986385749751715","date":"2025-08-26T16:05:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101787969740789778981646893005629542044","date":"2025-08-24T06:14:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T17:39:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103854545687850159594070784879257016200","date":"2025-08-17T09:59:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-14T14:31:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-31T12:56:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-24T11:02:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-20T09:41:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cardiovascular Disorders","date":"2025-07-20T09:38:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cardiovascular-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcar","sideBox":"Learn more about [BMC Cardiovascular Disorders](http://bmccardiovascdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcar/default.aspx","title":"BMC Cardiovascular Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6b8cfa6f-7e38-4f3e-9690-d6fc7caff7a8","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-08-22T13:11:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 13:11:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6635766","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6635766","identity":"rs-6635766","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}