Background
Type 1 diabetes mellitus (T1D) is a chronic T-cell–mediated autoimmune disease that results in the destruction of insulin-secreting β-cells [
1]. Diabetes is associated with multiple metabolic disorders that are characterized by hyperglycemia, which is accompanied by several complications [
2] that result from an absolute or relative deficiency in insulin secretion or action [
3]. Dyslipidemia is a common feature of diabetes, which is characterized by elevated triglyceride and low-density lipoprotein (LDL) cholesterol (LDL-C) levels [
4]. Hyperglycemia or dyslipidemia easily induces extensive oxidative stress that causes serious cellular dysfunction in diabetic patients [
5,
6]. Persistent hyperglycemia increases the production of free radicals, especially reactive oxygen species (ROS), in several tissues [
7]. Increased lipid peroxidation, characterized by increases in the levels of malondialdehyde (MDA), results in the formation of crosslinks between single molecules in proteins and the oxidation of LDL particles; oxidized LDL serves as the most common marker of oxidative stress [
8,
9].
Inflammation in autoimmune diseases is characterized by an imbalance between pro- and anti-inflammatory cytokines. Pro-inflammatory cytokines deleteriously influence insulin sensitivity and β-cell function [
10]. Interestingly, altered levels of cytokines impair insulin secretion in β cells [
11], and accumulating evidence supports that diabetes is a disease of the innate immune system [
11,
12]. Furthermore, diabetes increases the production of pro-inflammatory cytokines, including IL-1α, IL-1β, IL-6, and CXCL10 [
13,
14]. However, the main cytokines involved in diabetes pathogenesis are IL-1, TNF-α, and IL-6 [
15]. The impaired production of IL-1, IL-6, TNF-α and IFN-γ and the increased production of IL-10 in type 1 diabetic peripheral blood mononuclear cell (PBMC) cultures may indicate deficiencies in mononuclear cell activation and immune cellular adaptive responses [
16].
The increased incidence of infections in people with T1D is attributed to impairments in both humoral and cellular immune responses [
17]. Defects in CD8
+ CD28
+T suppressor lymphocyte populations have been identified in patients with T1D [
18]. Impairments in immune cells might interfere with normal pancreatic development and glucose homeostasis [
19]. In addition, defects in lymphocyte function have been suggested to contribute to disruptions in potassium channels [
20]. A previous investigation demonstrated that monocytes isolated from diabetic patients spontaneously secreted pro-inflammatory cytokines, leading to an altered T-cell response [
21]. We previously reported that the decreased proliferative capacity of lymphocytes contributed to the exhaustion of T cells during T1D [
22]. Chemokines play a crucial role in immune cell chemotaxis. In particular, CCL21 participates in naive T- and B-cell recruitment to the extra-follicular area in secondary lymphoid organs [
23]. CCL21 and CXCL12 are produced by cells scattered throughout the extra-follicular area and act via CCR7 and CXCR4, respectively, which are specifically expressed on activated T and B cells [
24]. The actin cytoskeleton is dynamically remodeled during B- and T-cell chemotaxis; this reorganization produces the force necessary for the activation and migration of these cells [
25].
Diabetic complications and immune response impairment are challenges in the clinical treatment of T1D; thus, the development of more effective treatment strategies is required. Propolis is a resinous natural material produced by bees from the collected exudates and buds of plants mixed with wax and bee enzymes [
26]. Propolis has several biological and pharmacological properties, such as immuno-modulatory, antitumor, anti-inflammatory, antioxidant, antibacterial and antiviral activities [
27‐
30]. However, the mechanisms by which propolis modulates the immune system during diabetes remain poorly understood. Therefore, the current study was conducted to investigate the direct effect of propolis supplementation on the impaired function of B and T lymphocytes during T1D.
Discussion
Natural antioxidants play central roles in enhancing the immune system via mechanisms that depend on oxidative stress; in turn, oxidative stress appears to play significant roles in many human diseases. In this context, we previously demonstrated that thymoquinone benefits the treatment of multiple myeloma and alleviates diabetic complications by restoring the T-cell immune response in diabetic offspring [
34‐
36]. Interestingly, we showed that natural antioxidants isolated from snake and ant venoms were able to enhance normal lymphocyte function and exert antitumor effects on breast cancer cells [
37,
38]. Propolis is a natural antioxidant product found in plant materials and is processed by worker bees. Natural antioxidants play various biological roles in the treatment of diabetic complications, including impaired wound healing [
39‐
41] and T-cell immune responses, in offspring born to diabetic mothers, as well as the treatment of other diseases, including cancer [
42‐
44]. Cytokines are essential mediators of intercellular communication that orchestrate the interactions of immune cells during immune responses. Thus, cytokine imbalances play a significant role in the acceleration of lupus-like autoimmune disease. Our study showed that oral supplementation of diabetic mice with propolis modulated glycemia by decreasing the blood glucose levels and increasing the insulin level to values similar to those observed in non-diabetic control mice. Moreover, treating diabetic mice with propolis improved the lipid profile and significantly inhibited oxidative stress by reducing lipid peroxidation and the free ROS levels in blood and in the liver and lymphoid organs. These improvements suggested that propolis acted as a strong anti-oxidant to ameliorate oxidative stress and delay the occurrence of diabetic complications. Our data are consistent with the results of a previous study, which reported that propolis significantly increased the plasma level of insulin [
45]. Furthermore, Fuliang et al. [
46] observed that the administration of propolis to STZ-induced diabetic rats may control glycemia and modulate glucose and lipid metabolism, leading to decreased release of lipid peroxidation products and increased free radical scavenging in diabetic rats.
Moreover, the oral administration of propolis extract significantly suppressed the blood glucose levels and helped to reduce dyslipidemia in diabetic rats [
47]. Propolis, which displays strong anti-oxidant activity, has been confirmed to suppress the MDA level and increase anti-oxidant activity in diabetic animal models and human patients [
48‐
50]. Diabetic complications are primarily attributed to increased ROS levels due to hyperglycemia [
51,
52]. Clinical trials also showed that improving oxidative stress may prevent the progression of both types of diabetes [
53,
54].
Importantly, T1D contributes to prolonged inflammation, which is characterized by the impairment of the immune response due to elevated levels of IL-1β, IL-6, and TNF-α [
55,
56]. Therefore, targeting inflammatory mediators has been proposed as an effective strategy to improve the immune response and modulate inflammation in diabetic patients. In the present study, we showed that propolis supplementation abrogated the inflammatory process associated with diabetes and restored the levels of IL-1 β, IL-6 and TNF-α to nearly normal levels. Propolis has been shown to directly inhibit cytokine production by immune cells [
57]. Khayyal et al. (2003) showed that the administration of an aqueous extract of propolis for the treatment inflammatory diseases decreased the levels of pro-inflammatory cytokines (TNF-α and IL-6) [
58].
In the present study, the plasma levels of IL-2, IL-4 and IL-7 were significantly reduced in diabetic mice, providing important evidence of impaired immune function. However, the IL-8 and IL-10 levels did not differ between diabetic mice and control mice or propolis-treated diabetic mice in this study. Furthermore, the plasma levels of IL-2, IL-4 and IL-7 were markedly decreased in diabetic mice, and these decreases were accompanied by a marked reduction in the proliferative capacity of antigen-stimulated B and T lymphocytes. Several studies have revealed that the plasma levels of IL-2, which promotes T-lymphocyte survival and proliferation, are consistently decreased in several diseases, indicating defective T-cell function. Furthermore, IL-7 plays complimentary roles in the maintenance of T cells after antigen stimulation [
59]. T cell survival may be impaired in the absence of IL-7 [
60]. Notably, the acute homeostatic proliferation of memory T cells has been shown to partly depend on the endogenous IL-7 levels [
61]. Additionally, IL-7 plays several important roles during B-cell development, including promoting the proliferation and survival of B-cell progenitors and the maturation of B cells during the pro-B- to pre-B-cell transition [
62]. Impaired T- and B-lymphocyte function has been linked to the abnormal activation of the immune system and has been shown to contribute to immunodeficiency [
63,
64]. In this study, we found that treating diabetic mice with propolis significantly restored both the levels of IL-2 and IL-7 and the proliferation of B and T lymphocytes. This restorative effect of propolis enhanced and maintained an efficient immune response by lymphocytes during T1D.
Chemokines play a crucial role in immune cell chemotaxis. CXCL12 and CCL21 participate in naive T- and B-cell recruitment to the extra-follicular area in secondary lymphoid organs via their lymphocyte receptors [
65]. Chemotaxis is an essential phenomenon for evaluating immune responses, and blocking chemokine receptors has recently been identified as a therapeutic strategy for various inflammatory and autoimmune diseases. Our data demonstrated that the percentages of chemotactic B and T cells were significantly reduced in diabetic mice but that treatment of diabetic mice with propolis significantly increased the percentages of chemotactic B and T cells. Supporting our results, previous studies reported that CD34+ cells isolated from diabetic patients demonstrated a marked defect in migration toward CXCL12 [
66]. Moreover, the CXCR4/CXCL12 signaling pathway has been shown to protect non-obese diabetic mice from autoimmune diabetes [
67]. Our results showed for the first time that propolis supplementation increased CCL21- and CXCL12-mediated B- and T-cell chemotaxis in diabetic mice. Previous studies have shown that CCR7 and CXCR4 are involved in the recruitment of blood-borne leukocytes to sites of inflammation [
68]. CCL21 and its lymphocyte receptor (CCR7) play a key role in the migration of lymphocytes from blood into lymphoid tissues [
69‐
71]. Recently, propolis has been shown to influence the immune system [
72,
73]. The immuno-modulatory activity of propolis has been shown to enhance innate immunity by activating the initial steps of the immune response via the upregulation of TLR-2 and TLR-4 expression, thereby contributing to lymphocyte activation by antigen-presenting cells [
73].
Competing interests
The authors declare that they have no competing interests, state that the manuscript has not been published or submitted elsewhere, state that the work complies with the Ethical Policies of the Journal and state that the work has been conducted under internationally accepted ethical standards after relevant ethical review.
Authors’ contributions
AA he was responsible for the extraction, preparation and characterization of the propolis. GB put the design of the study, carried out the immunological assays, prepared figures, drafted the manuscript and performed the statistical analysis. WNH put the design of the study, participated in the experiments and data analysis, prepared figures, drafted the manuscript. AA performed the biochemical analysis and participated in drafting the manuscript. NSA participated in the statistical analysis and drafting the manuscript. MAA participated in the analysis of data and drafting the manuscript. OG participated in the statistical analysis and drafting the manuscript. All authors read and approved the final manuscript.