Elevated Th17 and IL-23 in Hypertensive Patients with Acutly Increased Blood Pressure

Problem statement: Many severe cardiovascular complications such as st roke and heart attack can result from acutely increased blood pres sur in patients with hypertension. The underlying mechanisms remain unclear despite of extensive stud y. To characterize the inflammatory mechanisms in hypertensive patients with acute blood pressure increase, we tested circulating Th17 levels, IL-23R expression and plasma levels of IL-17 and IL-23 in hypertensive patients with acute blood pressure increase. Approach: 50 patients (24 males and 26 females) and 20 health y volunteers between the ages of 18 and 78 were enrolled in this study. 30 o f them were hypertensive patients with acute blood pressure increase (group A). The other 20 patients were hypertensive patients with steady blood pressure (group B). 20 healthy volunteers that were s xand age-matched to group B were recruited as normal controls (group C). IL-23R expression and Th 17 ratio in CD4 T cells were examined by Flow Cytometry (FCM). The levels of IL-17 and IL-23 in p lasma were measured using ELISA. Results: Increased Th17 and IL-23R CD4 T cells were detected in the patients of group A ( 1.4±0.6 and 8.6±3.3 respectively, p<0.05). No difference betwee n group B and C was observed (p>0.05). The levels of IL-17 and IL-23 in group A were significa ntly higher than group B and C (11.9±5.1 v.s. 7.8±3.8 v.s. 6.0±1.1 for IL-17, 3017±950 v.s. 2143± 927 v.s. 1916±935 for IL-23, P<0.05). No significant difference between group B and C was de tect d. Furthermore, there were a positive correlation between Th17/CD4 T ratio and IL-17 level (r = 0.514, p<0.05) and a p ositive correlation between IL-17 and IL-23 level (r = 0.837, p<0.05) i n all subjects. Conclusion: Our results suggested that IL-23 and Th17 cells may be involved in the pa thogenesis of acute blood pressure increase in the patients with hypertension. Understanding its infla mmatory characterization might be of fundamental importance for the prevention and treatment of acut e blood pressure increase in hypertensive patients.


INTRODUCTION
As a chronic medical condition characterized by elevated blood pressure, hypertension is recognized as a major risk factor for a variety of life threatening diseases including stroke, myocardial infarction, heart failure and aortic aneurysm. And most of these severe complications occur in the hypertensive patients with a sudden increase of blood pressure. Despite of extensive study, the pathophysiology of the sudden/acute increase of blood pressure in hypertensive patients is not clearly characterized. The involvement of immune activation in hypertension has been well demonstrated by many research groups (Guzik et al., 2007;Schmid-Schonbein et al., 1991;Dorffel et al., 1999). Th17, a recently discovered subset of CD4 + T cells, is believed to play a role in the pathogenesis of vascular dysfunction and hypertension (Madhur et al., 2009), In the current study, demonstrated an increased level of Th17 and IL-17 in hypertensive patients with acute increases of blood pressure, which is probably caused by the increased plasma IL-23 level and increased expression of IL-23R on CD4 + T cells. The characterization of its pathophysiology might be beneficial for the prevention and therapy of the sudden/acute increase of blood pressure in hypertensive patients.

MATERIALS AND METHODS
Subjects: 50 primary hypertensive patients including 24 males and 26 females, aged from 18-78 years, were recruited for this study. The patients were recruited from September 2010-February 2011. The patients were divided into two groups according to the symptoms of blood pressure: Group A, 30 patients with acute increase of blood pressure; Group B, 20 patients without acute increase of blood pressure. In addition to the 50 patients, we also included 20 healthy volunteers with normal blood pressure as control (group C).
Inclusion criteria for hypertensive patients with acute increase of blood pressure: (1) Onset of hypertension, SBP ≥ 160 mmHg and/or DBP ≥ 100mmHg, no previous history of hypertension found; (2) Blood pressure controlled in the normal course suddenly rise, SBP ≥ 160 mmHg and/or DBP ≥ 90mmHg; (3) Comply with the 2010 guidelines for hypertension prevention and treatment of hypertension in China definition and classification standards.
Specimen collection and cytokine detection: 5 ml venous blood was drawn from every patient after over 8 h fasting. Blood was collected in heparin anticoagulation tube. The blood samples of group A were collected within 24 h of acute blood pressure increase. Peripheral blood mononuclear cells were isolated for flow cytometry test. The plasma was separated from peripheral blood by centrifuging at 2000 rpm for 20 min. The levels of IL-17 and IL-23 in plasma were detected by corresponding ELISA kit (eBioscience, USA) as instructed by the manufacturer.
Statistical: All of data were analyzed by software SPSS17.0. The results were expressed as Mean ± SD, Comparison between multiple experimental groups was accomplished by one-way ANOVA with a post hoc test of LSD. p<0.05 was considered statistically significant. Correlation analysis was accomplished by using Pearson test.

The basic information and clinical data of subjects:
The basic information (age, gender, body mass index) and clinical data [blood pressure, cholesterol (TC), High Density Lipoprotein (HDL) and Blood Sugar (BS)] of the three groups of subjects are listed in Table  1. No significant differences were detected except for Systolic Blood Pressure (SBP) and Diastolic Blood Pressure (DBP). The SBP and DBP of group A were higher than group B and group C (p<0.01). There was no significant difference between group B and group C in blood pressure (p>0.05). Table 2 and Fig. 1-3, the ratio of Th17/CD4 + of group A was significantly higher than group B and group C (p<0.05); There was no significant difference between group B and group C (p>0.05); The ratio of CD 4 + IL-23R + /CD 4 + of group A were significantly higher than group B and group C (p<0.05). There was no significant difference between group B and group C (p>0.05); the ratio of CD 4 + IL-17 + IL-23R + /CD 4 + of group A were significantly higher than group B and group C (p<0.05). There was no significant difference between group B and group C (p>0.05).

The levels of IL-17 and IL-23 in plasma:
The plasma levels of IL-17 and IL-23 of group A were significantly higher than group B and group C (p<0.05). There was no significant difference between group B and group C (p>0.05) ( Table 3).
The correlation between IL-17 and IL-23: There were significantly positive correlations between the level of IL-17 and IL-23 in three groups ( Fig. 4-6). There was significantly positive correlation between the level of IL-17 and IL-23 in all subjects (r = 0.837, p<0.05; Fig. 7).
The correlation between Th17/CD 4 + T and IL-17: There were significantly positive correlations between the ratios of Th17/CD4 + T and the levels of IL-17 in all subjects (r = 0.514, p<0.05; Fig. 8).

DISCUSSION
Studies have shown that immune system plays a critical role in the development and progression of hypertension. Increased level of circulating cytokines and their ligands was detected in patients with primary hypertension (Eriksson et al., 2001;Virdis and Schiffrin, 2003). Higher numbers of activated mononuclear cells and lymphocytes were found in spontaneously hypertensive rat especially in adult rats (Schmid-Schonbein et al., 1991;Dorffel et al., 1999). Consistenly, the activation of inflammatory cells is also found in the peripheral blood of hypertensive patients (Dorffel et al., 1999). Inflammation and immune response, mediated by T lymphocyte can directly lead to vascular remodeling and increase of blood pressure (Schiffrin, 2010). Guziks have proven Th1 plays an important role in the pathogenesis of Ang II/DOCAsalt-induced hypertension and Th1/Th2 imbalance leads to hypertension (Guzik et al., 2007). Th17 is a recently discovered subgroup of helper T cell characterized by the secretion of IL-17. It is believed that Th17 may play a role in the pathogenesis of hypertension. However, its underlying mechanism is still unknown. In addition, it is unclear whether Th17 plays a role in the pathophysiology of acute blood increase within hypertensive patients, a common trigger for various severe cardiovascular complications. In this study, we found that the number of Th17 and plasma level of IL-17 in hypertensive patients with acutely increased blood pressure are significantly higher than that of both hypertensive patients without acutely increased blood pressure and healthy controls. Correlation analysis showed that Th17 cell percentage in peripheral blood cells is positively correlated with plasma IL-17 level. We failed to detect a statistical difference of Th17 and IL-17 between hypertensive patients without acutely increased blood pressure and healthy controls, despite of a slightly higher level in hypertension group. This is probably because the blood pressure in hypertensive patients without acute increase blood pressure was controlled to a similar level as healthy controls. Therefore we believe that Th17/IL-17 is one of the inflammatory mediators in the development and progression of essential hypertension.
In our study, acutely increased blood pressure group contains some incipient essential hypertension patients and some patients with previously controlled blood pressure but with a sudden increase of blood pressure. What they have in common is the sudden increase of blood pressure in a short time. Various factors can cause the sudden increase of blood pressure, such as weather changes, agitation, nervousness, fatigue and poor sleep. There are also some patients without any incentives. Based on the previous findings (Chen et al., 2004), we speculated that lymphocytes and monocytes are activated by AngII or other stimuli. The activation of those inflammatory cells results in an increase of Thl7 cells which in turn cause vascular inflammation and endothelial dysfunction by the secretion of IL-17, IL-6andTNF-α. The vascular inflammation and endothelial dysfunction finally lead to the increase of blood pressure. The elevation of blood pressure may also lead to endothelial damage which in turn induces inflammation and exacerbates hypertension. This may also explain why some essential hypertension patients who have controlled their blood pressure by taking antihypertensive drugs can have acutely increased blood pressure. We observed these patients and found that the same changes mentioned above. If we could break one of the above mentioned steps, the blood pressure might return to the stable stage again.
IL-23 is a new member of IL-12 family and mainly secreted by mononuclear macrophages and dendritic cells. It consists of a unique p19 subunit and a p40 subunit shared with the IL-12. IL-23 R is a new erythropoietin receptor family member and only expressed in the activated or memory cells.
Signaling through IL-23R, IL-23 activates STAT3 signal pathway and promotes the proliferation and migration of memory CD4 + T cell. It is an important factor in Th17 cell survival and proliferation (Aggarwal et al., 2003). In this study, IL-23R expression and the level of IL-23 in group A were higher than group B and group Candthere was no significant difference between group B and group C. It has been shown that IL-23R and IL-23 are involved in the pathogenesis of hypertension.
Increased blood pressure activated monocytemacrophage which secreted more IL-23. IL-23 in turn enhanced the cytokine secretion and antigen presentation of monocyte-macrophage and dendritic cell (Bastos et al., 2004). IL-23 plays an important role in the differentiation and proliferation of Thl7 cell through its binding to the IL-23R on T-cell. In this study, we found a positve correlation between IL-23 and IL-17 expression, indicating IL-23 might participate in the increase of blood pressure through promoting the differentiation of Thl7. Therefore, inflammation axis of IL-23/Th17/IL-17 is closely associated with the change of blood pressure and may be involved in the reoccurrence of hypertension.

CONCLUSION
In the current study, We demonstrated that IL-23 and Th17 cells may be involved in the pathogenesis of acute blood pressure increase in the patients with hypertension.
Understanding its inflammatory characterization might be of fundamental importance for the prevention and treatment of acute blood pressure increase in hypertensive patients.