Change of Peripheral Blood Treg/Thl7 in Cognitive Impairment with Chronic Renal Failure Patients

The aging of the global population has been associated with a gradual increase in high blood pressure, obesity and other risk factors, as well as increased morbidity due to chronic renal failure (CRF). This has imposed a heavy burden on families and has now become a global public health problem. A large-scale cross-sectional study in China showed a prevalence of chronic kidney disease (CKD) of 10.8%, for an estimated 120 million patients with this disease [1]. These patients may eventually progress to the CRF stage. Patients with CRF are prone to cognitive impairment, depression and other mental illness. About 1/3 of patients with CKD and end-stage renal disease have cognitive dysfunction.

Cognitive impairment has been positively correlated with glomerular filtration rate (GFR) and disease progression [2]. Cognitive dysfunction, which can include the expression of indifference or irritability, transient and delayed memory impairment, directional disorder, executive function decline and slow thinking, can seriously affect the patient’s daily life and learning work and reduce patient survival and quality of life. Some studies have shown that about 70% of patients with CRF undergoing dialysis have moderate or severe cognitive impairment [3]. Other studies have shown that CKD is independent of age, diabetes and high blood pressure or other risk factors leading to cognitive decline. However, the mechanism by which CKD promotes cognitive dysfunction in patients is unclear. Recent studies have found that inflammation and autoimmune system dysfunction may be associated with impairment of CRF cognitive function [4, 5].

Some immunoregulatory functions are controlled by a group of special T cell subsets, the CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells. These cells account for 5–10% of the peripheral CD4⁺ T cells and are responsible for inducing and maintaining immune homeostasis and resistance [6]. Regulatory T cells can be divided into natural Treg (nTreg) cells and inducible Treg (iTreg) cells, and they express CD25, CD39, CTLA-4 and Foxp3, as well as other molecules [6]. NTreg cells can significantly inhibit the proliferation of dendritic cells (DCs), monocytes, CD4⁺ T cells and CD8⁺ T cells by direct modulation of CTLA4 and other cell membrane inhibitory molecules that are in direct contact with effector cells. Treg cells play an important role in maintaining immune homeostasis and inducing immune tolerance primarily by secretion of IL-10, IL35, TGF-β and other anti-inflammatory factors [7]. Abnormalities in the number and function of Treg cells seriously affect the immune homeostasis of the body and are closely related to disorders such as autoimmune diseases, tumours and infections. Treg cell dysfunction plays an important pathological role in autoimmune diseases, chronic inflammation, infection, cancer and other diseases.

Th17 cells are one of the important helper T cell subsets identified in recent years. They promote inflammation in the body through the secretion of IL-17, IL-23, IL-6 and other cytokines, and are closely associated with autoimmune diseases, chronic infection and tumours [8]. The Treg/Th17 balance plays a key role in maintaining the stability of the immune state [9, 10]. An imbalance can cause a series of immune inflammatory responses that are characteristic of cancer, autoimmune diseases, atherosclerosis, allergic asthma and diseases of the development process [11-15]. Clinical studies have found that the presence of Treg cells in patients undergoing dialysis for end-stage renal failure significantly reduced the frequency of cellular and functional damage and significantly increased the Th17 cell frequency [16]. However, no study has yet determined an association between a Treg/Th17 imbalance and cognitive dysfunction.

The aims of the present study were therefore to investigate the changes in Treg/Th17 cell frequency and their corresponding cytokines in peripheral blood of patients with both CRF and cognitive impairment and to explore the potential role of Treg/Th17 in CRF-associated cognitive impairment.

A total of 71 elderly patients with CRF who had not undergone blood purification were enrolled in the hospital from January 2015 to October 2016. The degree of renal impairment was consistent with the National Kidney Disease Foundation’s “Kidney Disease Quality of Life Guidance” (K/DOQI) Phase 5 diagnostic criteria. The patients were 45 males and 36 females with an average age of 64 ± 8 years. Basic kidney diseases included 27 cases of chronic glomerulonephritis, 20 cases of diabetic nephropathy, 17 cases of hypertensive nephropathy, 6 cases of polycystic kidney disease and 4 cases of obstructive nephropathy.

Inclusion criteria were middle age (45 to 80 years old), non-dialysis patients, renal dysfunction of K/DOQI No. 5, clear mind, no mental illness, education level of primary school and above; compliance, and upon the completion of the study, signed informed consent. A total of 30 healthy middle-aged subjects, who were matched for sex, age, educational level, family background and underlying diseases, were selected as the normal control group (NC group).

Exclusion criteria included age <45 years, previous depression, epilepsy and other psychological or mental illness; major surgery 3 months before the study, trauma, use of antibiotics or immunosuppressive agents within 1 month before the study, illiteracy, severe hearing impairment or language communication disorders, a history of drug addiction or severe alcohol addiction, malignancy, severe cardiovascular or cerebrovascular diseases and liver disease.

Patient data: Medical records were utilised to obtain each patient’s age, sex, education years, levels of serum creatinine, urea nitrogen, haemoglobin, albumin, serum potassium, blood calcium, blood phosphorus and other biochemical indicators, and to calculate the creatinine clearance rate.

Treg and Th17 cell collection: The Treg/Th17 balance was determined from sterile elbow vein blood (8mL, EDTA anticoagulant tubes) obtained after fasting. Two tubes were collected, one for Treg/Th17 cell determination and the other tube for cytokine testing. The peripheral blood mononuclear cells (PBMCs) were extracted and the cell density adjusted to about 1×10⁶ cells/mL. For the Th17 test, 1 mL cell culture medium was centrifuged with 1640 medium containing 10% foetal bovine serum to resuspend the cells. A cell stimulation cocktail (eBioscience, USA) (2μL) was added, and the cells were transferred to a 24-well culture plate and incubated at 37 °C for 4 to 6 hours. The cells were washed with PBS and a cell membrane CD4-FITC antibody was added. The cells were fixed and then lysed after adding an intracellular anti-IL17-PE antibody.

For CD4⁺CD25⁺Foxp3⁺ Treg detection, 2mL of cells were centrifuged and then anti-CD4-FITC and anti-CD25-APC were added. The cells were lysed and an intracellular anti-Foxp3-PE antibody was added. The corresponding isotype control antibody was used as a negative control. Flow antibodies were purchased from Biolegend. Flow cytometry was conducted on a Model PC500MPL instrument (Beckman USA), and FlowJo7.0 software was used to analyse the flow results.

Detection of cytokines: Immuno-turbidimetric determination of C-reactive protein (CRP), ELISA detection of plasma IL-17, IL-10, IL-6 and TGF-β, detection kit were purchased from the United States Ebioscience company, all operations are strictly in accordance with the instructions.

Cognitive function assessment of patients: Patients were assessed in a quiet environment using a simple mental state assessment scale (Mini-Mental State Examination, MMSE). The scale included a total of 30 items and provided a comprehensive determination of space-time orientation, instant memory, attention and computing power, short-term memory, naming, ability to repeat, reading comprehension, language understanding and expression skills, and the total MMSE score was recorded. Patients with an MMSE total score <10 points were eliminated to avoid interference with floor effects. The values were revised according to education level, as follows: education years <6 years was scored 20 points, while education years> 7 years was scored 24 points; these values were lower than or equal to the threshold for cognitive dysfunction.

SPSS17.0 statistical software was used for statistical processing. Data were expressed as mean ± standard deviation (x ± s); the t-test was used for comparison between the two groups, and linear correlation analysis was used to determine the correlation between indicators. A value of P <0.05 indicated a statistically significant difference.

In this study, the 71 patients with CRF included 32 patients with cognitive impairment (45.1%), whose MMSE score was significantly lower than that of the CRF group with normal cognitive function or of a normal control group (P <0.05, Table 1). The levels of serum creatinine and urea nitrogen were significantly higher in patients with both CRF and cognitive impairment than in normal subjects. The levels of haemoglobin and creatinine were significantly lower in the CRF cognitive impairment group than in patients with CRF but no cognitive impairment (P <0.05, Table 1).

Fig. 1 shows that the frequency of CD4+CD25+Foxp3+ Treg cells in the peripheral blood (5.57±1.3%)was significantly lower in the CRF cognitive impairment group than in the CRF group with normal cognitive function (7.5 ± 0.9%) and normal control group (9.7 ± 1.7%). Fig. 2 shows that the frequency of Th17 cells (3.3 ± 0.7%) was significantly higher in the CRF cognitive impairment group than in the CRF group with normal cognitive function (2.2 ± 0.5%) and in the normal control group (1.5 ± 0.3%). We further confirmed these results using RT-PCR to determine the mRNA levels of RORγt, Foxp3, other markers of Tregs (CTLA-4, GITR and Helios) and HIF1α analysis in PBMCs obtained from the patients. The markers of Tregs were significantly reduced in all groups, the markers of Th17 RoRgt were markedly increased (Fig. 3), but HIF1a expression did not differ between the CRF group with cognitive impairment and the CRF without cognitive impairment. Real time RT-PCR primer sequence Table 2.

Fig. 4 shows a statistically significant difference (P < 0.05) in the levels of IL-6 (21.3 ± 5.1 pg/mL), IL-17 (18.5 ± 4.2 pg/ mL) and CRP (20.3 ± 5.9 mg/L) in the CRF group with cognitive impairment when compared with the CRF group with normal cognitive function (12.2 ± 4.5 pg/mL, 12.1 ± 3.7 pg/mL and 13.5 ± 4.6 mg/L, respectively) or the normal control group (9.2 ± 5.8 pg/mL, 7.4 ± 2.6 pg/mL and 3.2 ± 1.3 mg/L, respectively). The levels of anti-inflammatory factor IL-10 (7.4 ± 4.2 pg/mL) (13.8 ± 3.9 pg/mL), (18.3 ± 3.2 pg/mL) (P < 0.05), TGF-β (335.6 ± 175.3 pg/mL) were significantly lower than those in CRF cognitive normal group (512.7 ± 4.6 pg/mL), (953.8 ± 373.4 pg/mL), the difference was statistically significant (P < 0.05).

Correlation analysis showed that the MMSE score of patients with CRF was positively correlated with the frequency of peripheral blood Treg cells (r = 0.518, P< 0.05), but negatively correlated with frequency of Th17 cells (Fig. 5) (r = -0.435, P < 0.05).

In-depth immunological research has revealed new T cell subsets, such as Th9, Th17, Th22, Tfh and Treg [17]. Regulation of the balance between these T cell subsets and the production of a moderate immune response are conducive to maintaining the body’s immune home-ostasis and tolerance. Disorders of the Treg/ Th17 immune balance are closely related to diseases such as systemic lupus erythematosus, rheumatoid arthritis, atherosclerosis, chronic kidney disease, asthma and cancer. However, changes in the Treg/ Th17 ratio in patients with both CRF and cognitive dysfunction have not been previously evaluated. The present findings indicate that the frequency of Treg cells was significant reduced in the peripheral blood of patients with both CRF and cognitive impairment when compared with patients with CRF but with normal cognitive function. The corresponding Treg cytokines (IL-10 and TGF-β) were significantly decreased, whereas the frequency of Th17 cells and the corresponding cytokines (IL-17 and IL-6) were significantly increased in the patients with CRF and cognitive impairment. Correlation analysis showed that the MMSE score of patients with CRF was positively correlated with the frequency of Treg cells and negatively correlated with the frequency of Th17 cells in the peripheral blood. Therefore, the Treg/Th17 balance may play an important role in the cognitive impairment process in patients with CRF.

The decline in cognitive function affects many aspects of executive function, verbal memory, visual spatial skills and attention. Several scales, including the MMSE, the Montreal Cognitive Scale (MoCA), the Addenbrooke Modified Assessment Scale (ACE-R) and the Dementia Rating Scale (DRS), have been used to evaluate patients’ cognitive function [18, 19]. The MMSE scale has the advantages of rapidity, ease of operation and objectivity, and it can evaluate the sensitivity of the patients’ spatial and temporal orientation, memory, language expression and reading comprehension. It has a sensitivity of 80–90% and a specificity of 70–80%, so it is the most commonly used cognitive screening scale in the world [20].

The MMSE scale focuses on assessment of the language function of the candidate and is therefore susceptible to the patient’s level of education, but no significant differences were determined in the educational level of the members of the three groups in the present study. In the present study, the incidence of cognitive impairment was 45.1%, which was significantly lower than that of elderly patients with CRF, but our findings are in agreement with those of Witko-Sarsat [4] and Weiner [17]. The age, duration of illness, creatinine, urea nitrogen and haemoglobin levels and creatinine clearance were significantly different in patients with both CRF and cognitive impairment than in the CRF group with normal cognitive function. This suggests that early treatment of primary disease and complications can protect renal function and reduce cognitive impairment. Previous studies have found a positive correlation between the severity of cognitive dysfunction and the severity of renal dysfunction [21, 22]. We analysed this striking correlation further in the present study and found a positive correlation between cognitive impairment and CKD severity.

The observation that patients with CKD are prone to cognitive impairment is not surprising because CKD shares many of the same risk factors, such as age, hypertension, diabetes, vascular endothelial dysfunction, anaemia and lowered albumin [23]. In addition, cerebrovascular risk factors may lead to cognitive impairment in patients with Alzheimer’s disease or other neurodegenerative diseases, and they may play a critical role in cognitive impairment in patients with CRF [24]. A previous study reported that rapid deterioration of eGFR is closely related to cognitive decline and dementia [25]. Other vascular risk factors, such as hyperhomocysteinaemia, hypercoagulable state, inflammation and oxidative stress, are also associated with cognitive impairment [26]. These factors can accelerate the progression of atherosclerosis and vascular endothelial dysfunction, and both can increase the risk of dementia. In addition, a variety of uremic toxins cause cerebrovascular endothelial dysfunction, promote cognitive impairment, and lead to persistent deterioration of renal function [27-29]. Therefore, brain cognitive function and renal function appear to share a close and very complex pathophysiology. Cognitive disorders in patients with CRF may be associated with increased susceptibility to uremic-related toxins in brain tissue. Further in-depth study of the association between renal function damage and brain function in patients with CRF could help to reduce the risk of future cognitive impairment.

Some studies have shown that a lack of Treg cells significantly increased renal ischemia-reperfusion injury and the infiltration of neutrophils and macrophages, and that the use of adoptive transfer or drug stimulation of Treg cells can reduce the ischemic renal inflammatory response to protect kidney function [30]. A Parkinsonian animal model confirmed that Treg cells can reduce the brain Th17 cell-mediated inflammatory response to protect the function of the substantia nigra striatum [31]. In the present study we measured the frequency of peripheral blood Treg cells and the levels of plasma IL-10 and TGF-β1 in patients with CRF. The numbers of Treg cells and their corresponding cytokines were decreased in the cognitive impairment group, and the frequency of Treg cells was positively correlated with the MMSE score, indicating that Treg cells may have a protective effect against the onset of cognitive disorders. The reason for the reduction in frequency of Treg cells is unclear, but some studies have found that accumulation of toxic substances in patients with CRF leads to cell activation and induces apoptosis [32].

Th17 cells originate from the initial Th cells or memory T cells and express IL-17, IL-22 and RORγt molecules to induce proinflammatory factors (e.g. IL-6 and TNF-α), chemokines (e.g. MCP-1 and MIP-2) and matrix metalloproteinases. These, in turn, recruit inflammatory cells, such as neutrophils, and promote autoimmune and inflammatory responses [7]. The Th17 cells associated with autoimmune diseases, tumours, organ transplantation and other diseases show abnormal expression of the IL-17 cytokine, suggesting that this cytokine may be involved in the development of these diseases [33]. One line of evidence comes from mouse models, where IL-17 knockout mice show symptoms of multiple sclerosis and collagen-induced arthritis [34, 35]. Other studies have shown that the frequency of Th17 cells and the levels of IL-17 are significantly increased in peripheral blood of patients with lupus nephritis, whereas Treg cells and TGF-β are significantly decreased, thereby implicating the Treg/Th17 balance in the developmental process that leads to lupus nephritis [36]. An amyloid beta-induced rat dementia model indicated that the brain hippocampus undergoes infiltration by a large number of Th17 cells that produce IL-17 and IL-22 inflammatory factors, causing brain damage and neuronal apoptosis [37].

The Treg/Th17 balance is also essential for maintenance of immune homeostasis in the body, and its imbalance may be associated with CRF-related cognitive impairment. In the present study, the numbers of Th17 cells and IL-17 levels increased in the peripheral blood of patients with CRF and cognitive impairment when compared with a normal group or with a CRF group without cognitive impairment, while the number of Treg cells and related IL-10 and TGF-β factors decreased. These results suggest that the Treg/Th17 balance is important in patients with both CRF and cognitive impairment and that Treg/Th17 imbalance may play a potential role in eliciting cognitive impairment in patients with CRF.

Previous studies [4] also found significant increases in inflammatory mediators IL-6 and CRP in CRF patients with cognitive dysfunction. Brain-specific transgenic mice overexpressing IL-6 also showed severe neuropathological damage and cognitive dysfunction, and a specific mechanism was proposed involving IL-6 activation of neurotoxic molecules, NO release, promotion of β-amyloid protein synthesis and β-amyloid deposition in the brain as important pathological features of dementia [38]. Similarly, β-amyloid activation of glial cells, the release of IL-1, IL-6, NO and other inflammatory factors, destruction of the blood-brain barrier, promotion of infiltration by neutrophils, T lymphocytes and other non-specific inflammatory cells creates a malignant cycle [39]. IL-6 is also an important regulator of the Treg/Th17 balance, as it promotes the differentiation of primary T cells into Th17 cells and inhibits their differentiation into Treg cells [40]. In short, Th17 cells, through the secretion of IL-17 and other inflammatory factors, elicit continued brain nerve immune inflammation, which leads to neuronal damage and increased CRF cognitive dysfunction.

To the best of our knowledge, this is the first clinical study to focus on the Treg/ Th17 balance in patients with both CRF and cognitive impairment. A Treg/Th17 balance disorder was detected in patients with CRF and cognitive impairment, suggesting that this balance plays an important role in CRF-associated cognitive impairment. Th17 cells and their inflammatory factors may be important causes of cognitive impairment in patients with CRF, whereas Treg cells regulate the immune response through the secretion of anti-inflammatory factors, such as IL-10, thereby alleviating cognitive impairment. Studying the relationship between Treg/Th17 ratios therefore provides a new direction for the study of cognitive impairment in patients with CRF and indicates a new target for the treatment of CRF-associated cognitive impairment.

Research reported in this publication was supported by Guangxi Science and Technology Plan 2017GXNSFAA198320.

The authors declare that there are no conflicts of interest.