High soluble uric acid levels in healthy subjects are accompanied by elevated peripheral lymphocytes

doi:10.21203/rs.3.rs-2503666/v1

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High soluble uric acid levels in healthy subjects are accompanied by elevated peripheral lymphocytes

https://doi.org/10.21203/rs.3.rs-2503666/v1

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Background: Studies have shown that uric acid (UA) crystals can induce immune responses in the body; however, the relationship between soluble UA and immune responses remains unknown.

Methods: We retrospectively analyzed and compared peripheral blood immune cells and cytokine levels in healthy individuals with high or normal serum uric acid (SUA) levels. We used soluble UA to stimulate peripheral blood mononuclear cells in vitro and examined lymphocyte subset counts and activation.

Results: The number of immune cells in healthy individuals with high SUA levels increased, with the most obvious increase being observed in lymphocytes, mainly in CD4⁺ T cells. Lymphocyte elevation was more pronounced in women, who showed increased CD19⁺ B cell counts. Concomitantly, IL-4 and IL-10 levels significantly increased in healthy individuals with high SUA levels. In vitro, soluble UA promoted the proliferation and activation of CD4⁺ T and CD19⁺ B cells.

Conclusions: Our study revealed that elevated soluble UA levels may cause peripheral lymphocytes counts increase and make healthy subjects in a Th2-dominant immune status.

soluble uric acid

healthy subjects

peripheral lymphocytes

immune responses

Owing to the improvements in living standards, hyperuricemia has become a major public health problem and currently, is the second most common metabolic disease after diabetes [1]. As soluble uric acid (UA) levels continue to rise, UA crystals form and deposit in renal tubules, joints, and soft tissues, causing organ and tissue damage and leading to various diseases, the most common of which is gout [2]. Curiously, gout has been observed in only 36% of patients with hyperuricemia. Most patients exhibit elevated soluble UA levels without deposited UA crystals [3–5]. Current research on UA has focused on UA crystals, ignoring the value and role of soluble UA. Therefore, the potential role of soluble UA in the human body requires further investigation.

UA is the end product of purine and purine-containing substances degradation [6]. At physiological pH, the UA in the body exists mainly in its soluble form [7]. In the early days of human evolution, soluble UA was broken down by uricase, which reduced the amount of soluble UA in the body. However, with the loss of uricase, the kidney became the major site for soluble UA excretion, although only 10% of soluble UA is excreted in the urine while the remaining 90% is reabsorbed [8]. This finding implies that soluble UA may be beneficial to the body. Clinical and epidemiological studies have shown that high soluble UA levels are related to neuroprotection against Parkinson's disease, multiple sclerosis, Alzheimer's disease, and amyotrophic lateral sclerosis. Previous research has also revealed activation of the immune system as a potential mechanism for soluble UA-induced neuroprotection [9–13]. However, the relationship between soluble UA and immune system has not been confirmed.

Soluble UA has been found to drive epigenetic reprogramming in innate immune cells, causing a sustained increase in IL-1β gene transcription along with a persistent inflammatory response and increased responsiveness to secondary stimulation [14]. A previous study showed that soluble UA increased the expression of Toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing 3, IL-1β, human leucocyte antigen DR isotype, and CD40 [15]. CD40 is an important immune cell mediator that connects innate and adaptive immunity and enhances antigen presentation to T cells by activating dendritic cells [16]. Moreover, soluble UA increased CD86 expression when co-cultured with primary bone marrow-derived dendritic cells, providing co-stimulatory signals for T cells to initiate the immune response [17]. Furthermore, depletion of soluble UA suppressed the immune response related to dying cells [18]. Therefore, soluble UA may have an association with immune cells, although this relationship remains inconclusive.

Herein, we retrospectively analyzed the association between the levels of serum uric acid (SUA) in the form of soluble UA and the expressions of immune cells and cytokines to illustrate the impact of soluble UA on the immune response. In addition, we performed in vitro experiments to explore whether high levels of soluble UA can influence the counts and stimulate the activation of lymphocytes (LY), thus promoting immune responses. Our study provides clinical data to determine whether soluble UA can enhance the immune response, as well as valuable clues for future studies on related mechanisms.

Patients and general information

We randomly selected 2057 healthy subjects physically examined at the First Hospital of Jilin University from July 2020 to September 2020 to conduct this retrospective study. All healthy individuals included in this study had no cancer, heart disease, diabetes mellitus, fatty liver, hypertension, or other inflammatory disease during the study period. Healthy subjects with dysglycemia and dyslipidemia were excluded from the study. Of the 2057 healthy subjects, 1942 were included in the analysis of the correlation between different SUA levels and immune cell counts, and 119 were included in the analysis of the correlation between different SUA and cytokines levels (four of which were from the 1942 cases). Of the 1942 cases, 167 were used to analyze the correlation between different SUA levels and peripheral blood LY subsets. The general information of the healthy subjects involved in our study is presented in Table 1. At the same time, we also randomly selected 92 patients with hypertension, 37 patients with diabetes, 36 patients with heart disease, 1204 patients with fatty liver disease, and 1477 patients with breast cancer to analyze the relationship between different SUA levels and peripheral blood LY among patients with comorbidities. Table 2 presents the general information of the patients with these diseases. This study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the First Hospital of Jilin University (Approval No. 2021 − 678).Informed consent was waived owing to the retrospective nature of the study.

Table 1

Clinical characteristics of healthy people.
Parameter (Units)	Immune cells					Cytokines
Parameter (Units)	Normal SUA group	High SUA group	P- values		Normal SUA group		High SUA group	P-values
Number of samples, n	1543	258	NA	98			16	NA
Females, n (%)	979 (63.5%)	77 (29.8%)	NA			38 (38.8%)	6 (37.5%)	NA
Age(years)	43.49 ± 11.47	41.99 ± 12.60	< 0.0001			50.69 ± 12.24	53.00 ± 8.38	0.470
SBP (mmHg)	115.22 ± 12.07	118.63 ± 13.49	< 0.0001			NA	NA	NA
DBP (mmHg)	70.80 ± 8.89	73.39 ± 8.34	< 0.0001			NA	NA	NA
AST (U/L)	19.46 ± 8.97	21.17 ± 7.00	< 0.0001			20.15 ± 5.09	24.64 ± 8.75	0.004
ALT (U/L)	17.55 ± 16.05	22.93 ± 14.70	< 0.0001			18.31 ± 8.17	24.51 ± 9.63	0.007
GGT (U/L)	19.54 ± 25.11	29.52 ± 24.70	0.0004			21.21 ± 13.46	43.45 ± 34.05	0.020
ALP (U/L)	56.66 ± 15.99	61.11 ± 19.08	< 0.0001			67.50 ± 19.03	69.21 ± 14.23	0.732
TG (mmol/L)	1.26 ± 0.71	1.84 ± 1.35	< 0.0001			1.06 ± 0.26	1.22 ± 0.36	0.032
TC (mmol/L)	4.98 ± 0.96	5.12 ± 1.04	0.109			4.48 ± 0.58	4.39 ± 0.46	0.589
HDL-Ch (mmol/L)	1.30 ± 0.28	1.21 ± 0.25	< 0.0001			1.28 ± 0.30	1.25 ± 0.34	0.666
LDL-Ch (mmol/L)	2.87 ± 0.75	3.08 ± 0.82	0.0005			2.59 ± 0.35	2.53 ± 0.55	0.643
Cre(µmol/L)	61.95 ± 13.31	72.98 ± 13.99	< 0.0001			71.16 ± 14.51	73.98 ± 17.87	0.487
Glu (mg/dL)	NA	NA	NA			5.19 ± 0.43	5.12 ± 0.77	0.751

Table 2

Clinical characteristics of patients with these diseases.
Parameter (Units)		Hypertension																Fatty liver					Breast cancer
Parameter (Units)		Normal SUA group	High SUA group	P- values			Normal SUA group		High SUA group	P- values				Normal SUA group	High SUA group	P- values			Normal SUA group	High SUA group		P- values		Normal SUA group	High SUA group	P- values
Number of samples, n	70		22		NA	31		6			NA		29		7		NA	758			446	NA	1175		302		NA
Females, n (%)	24 (34.3%)		2 (9.1%)		NA	5 (16.1%)		2 (33.3%)			NA		9 (31.0%)		1 (14.3%)		NA	306 (40.4%)			104 (23.3%)	NA	1175 (100%)		302 (100%)		NA
Age (years)	60.64 ± 11.69		61.95 ± 11.46		0.646	58.39 ± 11.53		62.33 ± 14.46			0.470		60.07 ± 11.58		57.57 ± 17.21		0.645	47.89 ± 10.80			43.84 ± 12.18	< 0.0001	51.73 ± 10.19		54.52 ± 10.99		< 0.0001
SBP (mmHg)	149.80 ± 18.29		149.80 ± 17.53		0.987	124.70 ± 9.92		126.00 ± 10.14			0.779		117.80 ± 10.71		118.30 ± 15.39		0.915	121.00 ± 10.87			121.70 ± 9.99	0.231	-		-		-
DBP (mmHg)	88.03 ± 11.25		86.86 ± 9.06		0.659	75.74 ± 6.36		70.00 ± 9.21			0.068		72.28 ± 7.82		68.86 ± 7.49		0.303	74.21 ± 8.23			75.83 ± 8.05	0.001	-		-		-
PWB (10⁹/L)	6.31 ± 1.78		6.51 ± 1.31		0.628	7.09 ± 2.04		8.32 ± 1.72			0.174		6.77 ± 2.04		6.86 ± 1.30		0.912	6.61 ± 1.56			6.92 ± 1.66	0.001	5.55 ± 2.27		5.78 ± 2.17		0.112
NE (10⁹/L)	3.72 ± 1.37		3.94 ± 0.93		0.483	4.41 ± 1.67		5.24 ± 1.03			0.250		3.92 ± 1.36		4.55 ± 1.43		0.282	3.84 ± 1.21			3.96 ± 1.31	0.100	3.38 ± 1.98		3.48 ± 1.86		0.436
MO (10⁹/L)	0.43 ± 0.14		0.48 ± 0.14		0.117	0.47 ± 0.16		0.43 ± 0.05			0.563		0.47 ± 0.15		0.48 ± 0.11		0.901	0.45 ± 0.14			0.47 ± 0.14	0.017	0.37 ± 0.18		0.39 ± 0.18		0.034
AST (U/L)	22.26 ± 7.13		22.90 ± 6.98		0.729	19.90 ± 5.27		22.82 ± 10.19			0.327		21.58 ± 5.28		21.87 ± 6.60		0.900	22.05 ± 10.38			25.57 ± 12.12	< 0.0001	28.62 ± 19.18		51.30 ± 161.8		< 0.0001
ALT (U/L)	20.90 ± 10.39		21.95 ± 14.10		0.729	19.66 ± 9.79		18.92 ± 11.19			0.879		19.10 ± 8.39		19.79 ± 7.39		0.844	25.21 ± 17.41			34.44 ± 24.89	< 0.0001	26.83 ± 21.21		38.55 ± 59.51		< 0.0001
GGT (U/L)	28.11 ± 23.08		48.93 ± 47.48		0.010	29.71 ± 27.32		33.24 ± 26.95			0.791		30.13 ± 23.97		20.97 ± 15.19		0.344	30.92 ± 28.73			43.80 ± 42.46	< 0.0001	43.13 ± 68.91		77.14 ± 185.6		< 0.0001
ALP (U/L)	62.74 ± 15.71		74.52 ± 20.90		0.011	66.66 ± 20.92		72.42 ± 25.22			0.583		67.74 ± 16.66		63.39 ± 11.13		0.517	62.88 ± 16.13			63.71 ± 16.31	0.391	96.81 ± 54.93		120.6 ± 151.2		< 0.0001
TG (mmol/L)	1.91 ± 1.96		2.14 ± 1.39		0.620	1.16 ± 0.32		2.28 ± 1.38			0.793		1.55 ± 0.95		1.13 ± 0.40		0.264	2.06 ± 1.46			2.55 ± 1.77	< 0.0001	1.82 ± 1.16		2.56 ± 1.72		< 0.0001
TC (mmol/L)	5.14 ± 1.27		5.13 ± 0.89		0.978	4.98 ± 1.25		5.02 ± 1.11			0.947		5.60 ± 1.13		4.72 ± 1.72		0.108	5.20 ± 1.07			5.41 ± 0.97	0.001	5.02 ± 1.00		5.34 ± 1.12		< 0.0001
HDL-Ch (mmol/L)	1.23 ± 0.31		1.12 ± 0.17		0.132	1.16 ± 0.32		1.15 ± 0.21			0.982		1.26 ± 0.24		1.23 ± 0.42		0.785	1.19 ± 0.26			1.17 ± 0.23	0.192	1.41 ± 0.34		1.29 ± 0.31		< 0.0001
LDL-Ch (mmol/L)	2.99 ± 0.89		3.148 ± 0.80		0.495	2.90 ± 0.98		2.92 ± 0.81			0.972		3.41 ± 0.84		2.77 ± 1.03		0.093	3.13 ± 0.81			3.34 ± 0.77	< 0.0001	2.86 ± 0.74		3.09 ± 0.80		< 0.0001
Cre (µmol/L)	83.62 ± 95.40		91.78 ± 26.66		0.694	66.61 ± 13.22		95.48 ± 50.34			0.007		70.29 ± 13.99		106.7 ± 52.42		0.002	66.09 ± 13.07			73.91 ± 14.49	< 0.0001	55.85 ± 8.81		64.16 ± 19.21		< 0.0001
Glu (mg/dL)	5.62 ± 1.30		5.96 ± 1.11		0.300		9.57 ± 2.53	9.74 ± 4.08			0.893	5.20 ± 0.64			5.14 ± 0.56		0.807	5.40 ± 1.06			5.37 ± 0.84	0.654	5.62 ± 1.41		5.84 ± 1.23		0.016

Notes

Data are shown as mean ± SD. Statistical significance was indicated by p values as p < 0.05. SBP, systolic blood pressure; DBP, diastolic blood pressure; AST, aspartate transaminase; ALT, alanine aminotransferase; GGT, γ-glutamyl transpeptidase; ALP, alkaline phosphatase; TG, triglycerides; TC, total cholesterol; HDL-Ch, HDL-cholesterol; LDL-Ch, LDL-cholesterol; Cre, creatinine; Glu, glucose.

Detection of SUA concentration

Fasting venous blood was collected once in the morning. After separating the serum, the SUA concentration of all individuals was detected using the uricase-peroxidase method with a commercially available kit (Beckman Coulter, Inc. Brea, USA) and measured using a Beckman AU5841 automatic chemistry analyzer (Beckman Coulter, Inc.). The reference range of SUA concentration in the First Hospital of Jilin University ranged from 150–360 µmol/L for women and 210–430 µmol/L for men. According to this range, > 360 µmol/L or > 430 µmol/L were defined as a high level of SUA in women and men respectively, and results below this range were considered normal SUA levels.

Detection of immune cells in peripheral blood

Whole blood was used to detect the number and percentage of immune cells, including peripheral white blood cells (PWBCs), LY, neutrophils (NE), and monocytes (MO). Counts and percentages were analyzed using a Sysmex XN-9000 hematology analyzer (Sysmex, Kobe, Japan). The NE-LY ratio was determined by dividing the NE count by the LY count.

Detection of LY subsets and cytokines

Peripheral blood LY subsets were tested using a FACS Canto II flow cytometer (BD Bioscience, USA) with a BD Multitest 6-colour TBNK agent, including CD45-PerCP-Cy5.5, CD3-FITC, CD4-PE-Cy7, CD8-APC-Cy7, CD19-APC, and CD16/CD56-PE (BD Bioscience, USA). The fluorescent beads in the absolute counting tube were used as an internal standard for the absolute counts of LY subsets. The absolute cell counts and percentages of LY subsets were analyzed using BD FACS Canto software. The gating strategy used to analyze the LY subsets is shown in Fig. 1. The serum levels of cytokines (including TNF-α, IL-2, IL-4, IL-6, and IL-10) were measured by BD FACS Canto II flow cytometry (BD Biosciences, USA) using a Cytometric Bead Array with a Human Th1/Th2 subgroup detection kit (Jiangxi Nuode Company, China) following the manufacturer’s instructions. Cytokine data were obtained using the BD Diva software (BD Biosciences, USA). The flow cytometry was conducted daily using standard and CS&T application settings to verify the comparability of the machine performance and results.

In vitro experiments

In the in vitro experiments, we used Ficoll density gradient centrifugation (Ficoll-Histopaque-1077; Sigma) to separate and purify human heparinized peripheral blood and to isolate peripheral blood mononuclear cells (PBMCs). We aspirated 1000 µL of whole peripheral blood and diluted it with 1000 µL of phosphate-buffered saline. The diluted liquid was slowly dropped onto the surface of a 1500 µL Ficoll separator and centrifuged at 450 × g for 15 min (the speed of centrifugal ascent and descent was set to 1). After washing once with phosphate-buffered saline, the PBMCs (2× 10⁴ per well in a 96-well plate) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and stimulated with 714, 476, 238, or 95 µmol/L soluble UA at 37°C with 5% CO₂ (714 and 476 µmol/L were above the upper limit of the SUA reference value, 238 µmol/L was within the reference range, and 95 µmol/L was below the lower reference limit). After 18 h of culture, the PBMCs in each well were collected. The expression of CD45, CD3, CD4, CD8, and CD19 in the PWBCs was detected via surface staining with PerCP-labelled anti-CD45, FITC-labelled anti-CD3, APC-Cy7-labelled anti-CD4, PE-Cy7-labelled anti-CD8, and Apc-labelled anti-CD19 monoclonal antibodies for 15 min, followed by flow cytometry measurements. The results were analyzed by FlowJo software (version 10.0.7r2).

Statistical analysis

The Kolmogorov–Smirnov test (for number of cases > 50) or Shapiro–Wilk test (for number of cases < 50) was used to assess data distribution. When the data conformed to normal distribution and were described as means ± standard deviation, differences between two groups were compared using an independent samples t-test or paired t-test. Differences between multiple groups were compared using one-way ANOVA. When the data did not show normal distribution and were described as medians ± interquartile range, differences were compared using the Mann–Whitney U test. In this case, differences between multiple groups were compared using the Kruskal–Wallis test. Differences with P-values < 0.05 were considered significant. Data were analyzed using SPSS version 20 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA).

Relationship between different levels of SUA and immune cells in human peripheral blood

As SUA exists in the form of soluble UA, we used SUA instead of soluble UA to explore the potential link between different SUA levels and immune cells. We divided SUA levels into normal and high groups according to the SUA reference range and then compared the difference in the number of PWBCs, LY, NE, and MO and the percentage of LY, NE, and MO between the two groups. In the total population, the number of PWBCs in the high group was significantly higher than that in the normal group (P < 0.0001), and the same results were observed in the female population (P < 0.0001), but not in the male population (Fig. 2A). Importantly, in the overall population, although the numbers of LY, NE, and MO in the high group were higher than those in the normal group, the change in LY was the most significant, with a 12% higher increase in the high group compared with the normal group. Similarly, in the female population, the highest increase in the number of LY was observed in the high group, reaching almost 17% (Fig. 2B). No significant differences were observed in the percentages of LY between the two groups. Meanwhile, the percentage of MO was elevated in the high group, whereas the percentage of NE was elevated in the normal group (Fig. 2C). Therefore, we believe that SUA levels above the upper reference value limit may be mainly related to increased LY counts, which are more pronounced in the female population.

Increased peripheral blood CD4⁺ T cell number is related to high SUA levels

To further explore which type of LY was mainly associated with SUA, we compared the numbers and percentages of CD3⁺ T, CD4⁺ T, CD8⁺ T, CD19⁺ B, and CD16⁺CD56⁺ NK cells between the normal and high groups. Compared with the normal group, only the CD4⁺ T cell counts were significantly increased in the high group (P = 0.0019), whereas the percentage of LY subsets showed no difference between the two groups (Fig. 3A). Surprisingly, the number of CD4⁺ T cells was elevated in the female population, but the number of CD19⁺ B cells also increased significantly (P = 0.0142, P = 0.0284) (Fig. 3B). Meanwhile, the number and percentage of CD4⁺ T cells in the male subjects in the high group were higher than those in the normal group (P = 0.0199, P = 0.0375) (Fig. 3C). Hence, we believe that SUA levels above the upper limit in healthy subjects may enhance the number of CD4⁺ T cells, especially in women.

Increased IL-4 and IL-10 levels correlate with high SUA levels

To further explore the connection between different SUA levels and cytokines, we analyzed the changes in IL-2, IL-4, IL-6, IL-10, and TNF-α levels between the normal and high SUA groups. IL-4 and IL-10 levels were higher in the high group than in the normal group, and no significant changes were found in the levels of the other cytokines (P = 0.0254, P = 0.0019) (Fig. 4). These results revealed that healthy subjects with SUA levels above the upper limit of the reference value may exhibit a Th2-dominated immune environment.

Effects of stimulation of human PBMCs with soluble UA on LY counts

To determine the effect of SUA on LY counts, we isolated LY from fresh blood samples obtained from healthy donors and incubated them with soluble UA at concentrations of 714, 476, 238, 95, and 0 µmol/L for 18 h. The total number of LY cells and their subsets in the culture medium was determined using flow cytometry. LY was analyzed according to the gating strategy shown in Fig. 5A.

Compared with the soluble UA concentration of 0 µmol/L, soluble UA concentration of 714 µmol/L was associated with an increase in the counts of CD4⁺ T, CD19⁺ B, and CD19⁻ NK cells, and soluble UA concentrations of 476 and 238 µmol/L were associated with an increase in the counts of CD4⁺ T, CD8⁺ T, and CD19⁺ B cells. However, the difference did not reach statistical significance in LY treated with soluble UA at 95 µmol/L compared with that at 0 µmol/L (Fig. 5B). Notably, the number of CD19⁺ B cells stimulated by soluble UA was the highest and appeared to be dose-dependent, whereas other cells did not show the same trend (Fig. 5C). These results confirm that high levels of soluble UA in healthy individuals can cause an increase in LY counts.

Next, to explore whether soluble UA can activate LY, we analyzed the changes in LY size and CD45 expression under soluble UA stimulation. Intriguingly, we observed a large LY cell population where CD45 was highly expressed (Fig. 5D); therefore, we speculated that soluble UA might activate LY. After gating CD4⁺ T, CD8⁺ T, CD19⁺ B, and CD19⁻ NK cells from the CD45^highFSC^high fraction of LY (Fig. 5E), we compared the number of CD4⁺ T, CD8⁺ T, CD19⁺ B, and CD19⁻ NK cells in CD45^highFSC^high cells with or without soluble UA stimulation. Under stimulation with 476 µmol/L soluble UA, the counts of CD4⁺ T and CD19⁺ B cells among the CD45^highFSC^high cells significantly increased (P = 0.0228, P = 0.0187) (Fig. 5F). Therefore, we infer that higher levels of soluble UA in healthy individuals can induce the activation of CD4⁺ T and CD19⁺ B cells.

Relationship between SUA levels and LY counts in different diseases

To explore the specificity of SUA-induced LY proliferation, we compared the LY counts in the normal and high groups for different diseases. We found that high levels of SUA stimulated LY counts only in populations with fatty liver and breast cancer (P < 0.0001, P = 0.0026), and no difference was observed in populations with hypertension, diabetes mellitus, and heart disease (Fig. 6). These results suggest that high SUA levels can stimulate an increase in LY count in patients with fatty liver disease and in those with breast cancer.

The growing recognition that soluble UA plays a complex role in immune biology has stimulated strong interest in its relationship with immune cells. However, there are few existing analyses of the relationship between soluble UA and immune cells and even fewer domestic studies; therefore, our study is the first to combine retrospective analyses and in vitro experiments to investigate the effect of soluble UA on immune cells. We found that elevated soluble UA was mainly accompanied by an increase in CD4⁺ T cell count and IL-4 and IL-10 levels, implying that high levels of soluble UA may enhance the Th2-dominated immune response.

Studies in patients with hyperuricemia have shown that an increase in SUA concentration is related to signs of inflammation, such as an increase in the number of PWBCs [19]. This is in line with our clinical results. CD4⁺ T cells are divided into Th1 and Th2 cells, the latter of which secrete IL-4, IL-6, and IL-10 [20]. Thus, our findings indicate that when SUA is high, the cytokine environment of healthy subjects may shift towards Th2 cytokines, which is essential in humoral immunity. Therefore, we used different levels of soluble UA to stimulate PBMCs from healthy individuals in an in vitro experimental platform to verify our clinical results. The results showed that high levels of soluble UA can increase the CD4⁺ T and CD19⁺ B cell counts in healthy subjects. Moreover, since humoral immunity is mainly mediated by B cells, this further confirms our hypothesis that high levels of soluble UA may be accompanied by stimulation of humoral immunity. However, we need to analyze the relationship between soluble UA and levels of complement and antibodies to further explain our assumption.

In addition, studies have shown that enhanced Th2-dominated immune responses are mainly related to allergic and autoimmune diseases, which are more common in women [21, 22]. This may explain why the number of CD4⁺ T cells in women increased more significantly in our clinical results along with the increase in CD19⁺ B cells. Additionally, our study showed an increased number of LY in fatty liver and breast cancer patients with high levels of soluble UA. The findings match those observed in earlier studies. Research found that soluble UA can enhance the anti-tumor response of Th2 cells, promote antibody production by B cells, and relate to CD8⁺ T and NK cells [23]. Moreover, studies have confirmed that soluble UA as an adjuvant can play a role in the anti-tumor immunity induced by dendritic cell vaccines [24, 25]. Epidemiologic evidence has shown that elevated soluble UA concentrations were associated with the progression of hypertension, cardiovascular disease, and metabolic syndrome [26]. Therefore, we believe that soluble UA may be involved in the development of the disease by promoting LY proliferation to regulate the immune environment. However, these results were not observed in patients with heart disease, hypertension, and diabetes, probably due to the small sample size.

We found that both high levels and physiological concentrations of soluble UA can cause proliferation of LY. A possible explanation for this might be that any concentration of soluble UA can inhibit interleukin-1 receptor antagonist promoting TLR-induced proinflammatory cytokine IL-1β production by human primary cells [27]. IL-1β is a key mediator of the inflammatory response and induces the immune response of the body. Despite being essential for the host response and resistance to pathogens, it also exacerbates damage during chronic disease and acute tissue injury [28]. Therefore, soluble UA may promote an increase in LY population through the IL-1β signaling pathway. Since clinical data showed that LY numbers increase with elevated soluble UA, we suspected that physiological concentrations or higher concentrations of soluble UA may contribute to host defense mechanisms, and that high concentrations of soluble UA may lead to sterile inflammation and pathological consequences. However, in vitro experiments found no dose-dependent relationship between soluble UA and LY, probably due to the small sample size and interindividual differences. In the future, we plan to expand the sample size and further investigate the mechanisms by which soluble UA promotes LY proliferation.

High levels of soluble UA in healthy individuals were related to increased CD4⁺ T cell counts and IL-4 and IL-10 levels, suggesting that high levels of soluble UA may enhance the Th2-dominated immune response. Hence, physicians should pay attention to the potential clinical significance of the LY subgroup or Th1/Th2 cytokines in the hyperuricemic population. Besides, soluble UA also can be used as an immune adjuvant in the management of certain diseases treatment, opening a new perspective for therapeutics.

ALP Alkaline phosphatase

ALT Alanine aminotransferase

AST Aspartate transaminase

Cre Creatinine

DBP Diastolic blood pressure

GGT γ-Glutamyl transpeptidase

Glu Glucose

HDL-Ch HDL-cholesterol

LDL-Ch LDL-cholesterol

LY Lymphocytes

MO Monocytes

NE Neutrophils

PWBCs Peripheral blood white cells

SBP Systolic blood pressure

SUA Serum uric acid

TC Total cholesterol

TG Triglycerides

TLR4 Toll-like receptor 4

UA Uric acid

Acknowledgments

We would like to thank Editage (www.editage.cn) for English language editing.

Author contributions

QYY: conception and design of the work, analysis and interpretation of the data, and preparation of the first draft of the manuscript. ZYS: analysis and interpretation of the data, performed the interpretation of the imaging. YW and XD: performed the data collection. LYW. and JH: guidance of the research and revision of the manuscript. All authors read and approved the final submitted version of the manuscript to be published as presented.

Funding

No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.

Availability of data and materials

The datasets generated or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This was a retrospective study with clinical data collection, and First Hospital of Jilin University approved to waive the patient consent (Reference: 2021-678). This study was conducted according to the guidelines of the Declaration of Helsinki. Informed consent was waived owing to the retrospective nature of the study.

Consent for publication

All the authors consented to publish work.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Department of Laboratory Medicine, The First Hospital of Jilin University, Jilin University, Changchun, Jilin, P.R. China 130021. ²Institute of Pediatrics, The First Hospital of Jilin University, Jilin University, Changchun, Jilin, P.R. China 130021. ³Department of Molecular Biology, College of Basic Medical Sciences, Jilin University, Changchun, Jilin, P.R. China 130021

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No competing interests reported.

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High soluble uric acid levels in healthy subjects are accompanied by elevated peripheral lymphocytes

Status:

Version 1

Abstract

Figures

Background

Methods

Patients and general information

Detection of SUA concentration

Detection of immune cells in peripheral blood

Detection of LY subsets and cytokines

Statistical analysis

Results

Relationship between different levels of SUA and immune cells in human peripheral blood

Increased peripheral blood CD4⁺ T cell number is related to high SUA levels

Increased IL-4 and IL-10 levels correlate with high SUA levels

Effects of stimulation of human PBMCs with soluble UA on LY counts

Relationship between SUA levels and LY counts in different diseases

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

High soluble uric acid levels in healthy subjects are accompanied by elevated peripheral lymphocytes

Status:

Version 1

Abstract

Figures

Background

Methods

Patients and general information

Detection of SUA concentration

Detection of immune cells in peripheral blood

Detection of LY subsets and cytokines

Statistical analysis

Results

Relationship between different levels of SUA and immune cells in human peripheral blood

Increased peripheral blood CD4+ T cell number is related to high SUA levels

Increased IL-4 and IL-10 levels correlate with high SUA levels

Effects of stimulation of human PBMCs with soluble UA on LY counts

Relationship between SUA levels and LY counts in different diseases

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1

Increased peripheral blood CD4⁺ T cell number is related to high SUA levels