The delicate balance between pro- and anti-inflammatory cytokines determines the net effect of an inflammatory response. Perturbations in this equilibrium can drive the host defense immune response either towards chronic inflammation (pro-inflammatory) or towards healing (anti-inflammatory). Exposure of endothelial cells to pro-inflammatory cytokines leads to transient and reversible endothelial dysfunction [
105],[
106]. A number of anti-inflammatory treatment strategies improve endothelial function by preventing pro-inflammatory cytokine synthesis. Anti-inflammatory cytokines are a series of immune-regulatory molecules that control the pro-inflammatory cytokines response, which consequently reduces inflammation and promotes healing. In addition, an elevation in the level of anti-inflammatory cytokines can also be found in the development of vascular disease [
107], which reflects an early compensatory mechanism and serves as an indicator of pro-inflammatory reactions. Major anti-inflammatory cytokines include IL-1Ra, IL-4, IL-10, IL-11, IL-13 and TGF-β. Several newly found cytokines, such as IL-33, IL-35, and IL-37 also participate in regulating the function of EC. The following will discuss two of these anti-inflammatory cytokines including IL-10 and TGF-β in detail.
Interleukin-10
IL-10 is an anti-inflammatory cytokine produced by many types of immune cells, such as monocytes, macrophages, type 2 T helper cells (Th2), mast cells, natural killer (NK) cells, and CD4 + CD25 + Foxp3+ regulatory T cells (Tregs). Its primary biological function is to limit and terminate inflammatory responses and regulate the differentiation and proliferation of several immune cells. IL-10 receptor 1 (IL-10R1) and IL-10R2 are two subunits of the IL-10 receptor that are expressed by hematopoietic and nonhematopoietic cells. The receptor expression has also been observed in endothelial cells [
108],[
109], which provides the structural evidence for IL-10 to not only counteract pro-inflammatory cytokines but also potentially inhibit endothelial dysfunction directly. Many studies have found that IL-10 is a key mediator of vascular protection in atherosclerosis, type II diabetes and hypertension [
110]-[
112]. IL-10 is shown to protect endothelial function by initiating the degradation of several cytokine mRNAs, inhibiting the production of monocyte/macrophage- and neutrophil-derived cytokines [
113] and attenuating induction of superoxide generation within the vascular wall [
114],[
115]. Clinically, in patients with coronary artery disease, IL-10 serum level acts as an independent predictor of the endothelium-mediated vasodilator response of the forearm circulation [
116]. Furthermore, IL-10 prevents the impairment of endothelial dysfunction induced by elevated levels of C-reactive proteins [
116].
IL-10 also attenuates inflammatory responses by its antioxidant properties. It plays a protective role in blood vessels by inhibiting NADPH oxidase activity and ROS production [
111],[
117]. In addition, IL-10 can restore Ang II-induced endothelium-dependent relaxation impairment measured by wire myographs in healthy murine aorta rings [
118]. Mechanistically, Ang II leads to endothelial dysfunction by increasing gp91phox (NOX2) expression, which is a subunit of NADPH oxidase, while IL-10 inhibits this response by normalizing NADPH oxidase protein expression. In IL-10 knockout (IL-10
-/-) mice, carotid arteries and thoracic aortas show a marked augmentation of vascular dysfunction after systemic treatment with Ang II that can be prevented by the treatment with superoxide dismutase-mimetic compound TEMPOL [
112]. In Ang II-infused hypertensive mice, IL-10 that is released by transferred CD4
+CD25
+ natural Treg cells from wild type mice significantly reduces NAPDH oxidase activity and systolic blood pressure; while the transfer of Tregs isolated from IL-10
-/- mice has no effect on the hypertension mice. Collectively, these results suggest that IL-10 generated by the immunosuppressive Treg cells protects against Ang II-induced vascular dysfunction and hypertension development by suppressing oxidative stress [
119].
In recent studies on aging, old IL-10
-/- mice are shown to have stiffer vessels and more severe endothelium-dependent relaxation impairment than wild type mice, which can be reversed by a scavenger of superoxide [
120]. In addition, gp91phox is significantly induced in IL-10
-/- than that of wild type mice in aging vessels. Furthermore, it is demonstrated that IL-10 protects against age-related endothelial dysfunction by the inhibition of oxidative stress. Aside from oxidative stress, it was also found that there is an increase of COX-2 activity and consequently activation in thromboxane A
2 receptor instead of PGI
2 in older IL-10
-/- mice [
121].
In vitro, IL-10 suppresses the production of pro-inflammatory cytokines such as interferon-γ (IFN-γ
), IL-1β, TNF-α, and IL-6 by immune cells including T cells, monocytes, macrophages and dendritic cells to produce [
113],[
122]-[
124]. Meanwhile, IL-10 blocks the activity of NF-κB [
125], which is a key pro-inflammatory transcription factor [
126]-[
128].
In vivo, IL-10 dampens the pro-inflammatory effects of IL-1 and TNF by stimulating IL-1Ra and soluble TNF receptors (sTNFR) production [
129]. In TNF-α-treated mouse models, impairment of vascular relaxation is accompanied with a reduction of eNOS, which can be counteracted by IL-10 which induces eNOS expression and attenuates superoxide production [
130]. In addition, by using mouse aortic rings in a myograph, TNF-α triggers a significant decrease in ACh-induced relaxation, which can be restored by IL-10.
The Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway plays an essential role in mediating the anti-inflammatory actions of IL-10 [
131]. In human EC, IL-10 up-regulates eNOS expression and activity mediated by the activation of STAT3 [
132]. IL-10 and IL-10 receptor interaction involves the JAK family tyrosine kinases Jak1 and Tyk3 that induce tyrosine phosphorylation and the activation of latent transcription factors STAT3, STAT1, and STAT5 [
133],[
134]. IL-10 inhibits pro-inflammatory cytokine production in the macrophages via a JAK/STAT3-dependent pathway [
135]. In IL-10
-/- mice, STAT3 phosphorylation induction does not occur.
Aside from the JAK/STAT pathway, recent studies suggest that IL-10 also confers endothelial protection through several other signaling pathways. IL-10 inhibits the IL-1-induced inhibitor of kappa B (IκB) expression, decreases IκB phosphorylation and causes an increase in the eNOS activity [
136]. In addition, IL-10 is associated with the inhibition of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activity and the MAPK kinase (MEK)/ERK pathway [
137]-[
139]. In TNF-α-infused IL-10
-/- mice there is an increase of total and phosphorylated ERK1/2 [
140], and the aorta and mesenteric arteries isolated from those mice display increased contractile responses to ET-1 through the ET
A receptor, which can be abrogated by the ERK1/2 inhibitor PD-98059. This result demonstrates that IL-10 attenuates ET-1 induced vascular injury through the inhibition of the ERK1/2 pathway.
Emerging evidence also suggests that IL-10 plays a major role in suppressing endothelial dysfunction in lipopolysaccharide (LPS)-induced endotoxemia. In LPS-induced endotoxemia, activated ERK1/2 can induce higher expression of IL-10. IL-10 in turn restores eNOS-mediated relaxation by inhibiting production of ROS in monocytes and neutrophils. Meanwhile, it also decreases the production of pro-inflammatory cytokines such as TNF-α and IL-6, leading to an attenuation of septic shock [
141]. Mechanistically, IL-10 inhibits the transcription of several inflammatory genes that are induced by the Toll-like receptor (TLR) signaling, such as COX-2, IL-8, and IL-1 [
142],[
143]. This response can be completely or partially abrogated by the PI3K or Akt1/2 inhibitor. The PI3K-Akt-glycogen synthase kinase 3 (GSK3) pathway regulates IL-10-induced gene expression and controls the ability of IL-10 to suppress a set of inflammatory genes [
144].
In summary, as a cytokine synthesis inhibitor factor (CSIF), IL-10 inhibits a broad spectrum of the functions of activated monocytes/macrophages and T cells, including pro-inflammatory cytokine synthesis, and NO production. It also contributes to an essential part in the balance between pro- and anti-inflammatory cytokines.