Introduction

Breast cancer is the most common cancer among women, comprising about one-fourth of all female cancers worldwide [1, 2]. It is well-recognized that the functional status of immune system has direct bearing on breast cancer. But, the exact biological mechanism involved in breast cancer pathophysiology is still not clearly understood [3]. Numerous reports suggest that modulation of the innate and adaptive immune response through B cells, T cells, macrophages, dendritic cells (DC), natural killer (NK) cells, and other mediators is critical in initiation and progression of breast cancer. Role of immune system and inflammation in breast cancer has been extensively reviewed elsewhere [4, 5]. Cancer immunotherapy employs a variety of options, including enhancement of stimulatory signals required for T cell activation, genetically engineered cells to secrete cytokines to enhance the intensity of anti-tumor immune response, and direct exogenous therapeutic use of cytokines. Immunoregulatory cytokines are an important component of biological milieu associated with breast cancer [6]. Several cytokines including Interferon (IFN)-α, β, and γ; Interleukin-2 (IL-2), IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) are known to play important role in coordinated manner in breast carcinogenesis [7, 8].

IL-10, initially known as cytokine synthesis inhibitory factor, is primarily a potent anti-inflammatory cytokine that inhibits gene expression and T cell/macrophage cytokine synthesis and inhibits their antigen-presenting capacity [9]. It suppresses production of IL-1α, IL-1β, TNF-α, IL-6, IL-8, IL-12, IL-18, granulocyte–macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein-1α (MIP-1α), RANTES (Regulated upon activation, normal T cell expressed, and secreted), leukemia inhibiting factor, and IL-10 itself [9]. IL-10 also inhibits IFN-γ synthesis by activated Th-cells and peripheral blood mononuclear cells (PBMC) and induces mast cell proliferation [10]. IL-10 is also a strong stimulator of B cell differentiation for immunoglobulin secretion [9]. IL-10 inhibits nuclear factor-κB (NF-κB) translocation considered as a mechanism for inhibiting immediate-early pro-inflammatory response [11].

IL-10 is located on chromosome 1 and mature human IL-10 consists of 160 amino acids with molecular weight of approximately 18 kDa in monomeric form. The homodimeric protein with single transmembrane domain subunits binds to class II cytokine receptor [12]. Human IL-10 contains four exons which show 73% amino acid homology with murine IL-10 [10]. IL-10 structure, its receptor along with their role in physiological functions and pathological conditions like inflammatory diseases have been widely reviewed elsewhere [13–20].

IL-10 production and signaling mechanism

IL-10 is an acid-labile cytokine produced by almost all leukocytes [21], including T cells, Ly+1 B cells, monocytes, macrophages, and keratinocytes [10]. Major sources in vivo are mainly monocytes, macrophages and Th cells; but it is also secreted by DC, B cells, cytotoxic T cells, γδ-T cells, NK-cells, mast cells, as well as neutrophilic and eosinophilic granulocytes [20]. IL-10 secretion in defined situation is dependent on the kind of stimulus, type of affected tissue, and time point in an immune process [22].

IL-10 was originally described as a cytokine produced by Th2 cells, but later on it became evident that IL-10 is also produced by Th1 cells [23]. IL-10 activity is mediated by its specific cell surface receptor complex, which is expressed on a variety of cells, particularly in immune cells [19]. The IL-10/IL-10R interaction activates tyrosine kinases, Jak1 and Tyk2, which are associated with IL-10R1 and IL-10R2, respectively [15]. These kinases are responsible for the phosphorylation of tyrosine residues within the intracellular domain of IL-10R1 which serve as docking sites for Signal Transducer and Activator of Transcription (STAT) molecules [24]. The receptor engagement and tyrosine phosphorylation activates the cytoplasmically localized inactive transcription factors STAT-1, STAT-3, and STAT-5, resulting in translocation and gene activation [25]. IL-10R has a strong preference for STAT-3 and cause transcriptional induction via gene regulatory elements [26]. IL-10 rapidly activates STAT-3 and it remains phosphorylated over a sustained period. This is in contrast to IL-6 mediated STAT-3 activation which is transient [27].

IL-10 expression is regulated by the balance of STAT-3 and SOCS3 (Suppressor of Cytokine Signaling-3). In a recent study, STAT-3 silencing found to reduce IL-10 expression significantly, while SOCS3 silencing induced [28]. Recently, NRDG2 (N-myc downstream regulated gene 2) expression has been shown to modulate SOCS3 and STAT-3 activity, eventually leading to inhibition of IL-10 production [29]. Signaling mechanism particularly MAP-kinase pathways involved in anti-inflammatory action of IL-10 is reviewed by Haddad et al. [30]. Although our knowledge about IL-10 production and IL-10 mediated signaling is expanding, still many aspects remain unexplained.

IL-10 is also secreted by tumor cells [31]. Numerous human cancer cell lines have been shown to secrete IL-10 into their supernatant [32]. The level of expression was more in tumor and breast cancer cell co-culture environment [33]. Breast tumor cells express high level of IL-10 mRNA [34]. However, source of increased serum level of IL-10 in cancer patient is mostly tumor infiltrating suppressor macrophages instead of tumor cells. [35].

Effects of IL-10 on immune cells and cytokines production

IL-10 interacts with a number of cytokines, mostly causing inhibition. IL-10 primarily acts on DCs and macrophages, and inhibits antigen presentation [36, 37]. Haddad et al. [30] reviewed inhibitory action of IL-10 on various cytokines and possible signaling mechanism involved. Figure 1 depicts few known interactions of IL-10 with various immune and tumor cells.

Fig. 1
figure 1

Role of IL-10 relevant to breast cancer initiation, progression, as well as regression. Light colored arrow shows cells secreting IL-10, dark arrow shows cells stimulated by IL-10, dotted arrow shows differentiation T-shaped arrow shows process blocked by IL-10, and lightning arrow shows detrimental effect on tumor cells

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IL-10 inhibits major histocompatibility complex class II (MHC-II) expression as well as up-regulation of co-stimulatory molecules CD80 and CD86. IL-10 inhibits DC maturation and differentiation from monocyte precursors [38]. Thus, immuno-inhibitory properties of IL-10 is mainly due to their effect on antigen-presenting cells (APC) to prevent production of Th1 and Th2-associated cytokines [39]. IL-10 inhibits both proliferation of CD4+ T cells and production of IL-2 and IFN-γ by Th1 as well as IL-4 and IL-5 by Th2 [40]. IL-10 inhibits release of pro-inflammatory mediators from monocytes/macrophages and inhibits LPS (lipopolysaccharide)- and IFN-γ-induced secretion of TNF-α, IL-1β, IL-6, IL-8, G-CSF and GM-CSF [41]. IL-10 prohibits human monocytes from producing IL-1α, IL-1β, IL-6, IL-8, TNF-α, and G-CSF [42]. IL-10 also inhibits monokine synthesis more efficiently than same concentrations of IL-4 [43].

IL-10 acts as a co-stimulator for the proliferation of mast cells and peripheral lymphocytes. Optimal mast cell growth is achieved by a combination of IL-3, IL-4, and IL-10. IL-10 alone has no effect on mast cell proliferation [30]. IL-10 in humans exerts pro-inflammatory effects by enhancing IL-1 production [20]. IL-10 has also been shown to inhibit LPS-induced IL-1β production in human monocytes. IL-10 resulted in an increase in the ratio of IL-1RA to IL-1β in both neutrophils and monocytes [44].

Th17 cells are considered as developmentally distinct population from Th1 and Th2 cells and secrete IL-17. Recently, several studies reported interactive role of IL-10 and IL-17 suggesting importance of IL-10 signaling in direct inhibition of Th17 cells [45, 46].

IL-10 inducing and inhibiting agents

Anti-CD3 antibody and LPS are classically known to induce IL-10 production [47]. Similarly, IgA can also induce IL-10 expression in human monocytes [48, 49]. TGF-β and IL-6 induces IL-10 and IL-17 in normal conditions and IL-10 in turn helps in regulation of actions of IL-17 [50]. IL-27 and TGF-β induce IL-10 production and stimulate IL-10 producing cells [51, 52]. Stimulation with LPS, IL-1α or TNF-α weakly activates IL-10 expression [53].

Recently, CLA (conjugated linoleic acid) has been reported to induce IL-10 which in turn has an immunostimulatory effect on PBMCs via up-regulation of TNF-α production. But, IL-10 causes down-regulation of TNF-α production and exerts anti-inflammatory effect in LPS-stimulated PBMCs [54]. Another report showed G-1, a G protein coupled estrogen receptor (GPER) agonist and Thalidomide can induce IL-10 expression directly acting on Th17 or hybrid T cell populations [55]. Thalidomide also induces IL-10 expression in stromal population of bone marrow derived from patients with myelodysplastic syndromes [56]. Similarly, suppression of histone deacetylase 11 promotes IL-10 expression in Kupffer cells and induces tolerance following orthotopic liver transplantation in rats [57]. Apoptotic cells selectively affect IL-10 production induced by zymosan, a crude β-glucan used as fungal surrogate [58]. Transcription factors such as, cMaf also regulates IL-10 expression and T-effector development [59].

Several immunomodulators and other drugs are reported to inhibit IL-10. Immunosuppressive agent cyclosporin blocks IL-10 production [60]. 15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) inhibits IL-10 mediated activation of STAT3 and blocks IL-10 signaling [61]. AS101 (ammonium trichloro (dioxoethylene-o,o′) tellurate), an immunomodulator also inhibits IL-10 signaling [62]. Recently, agents other than immunomodulators are also reported to inhibit IL-10 production. For example, Rituximab inhibits IL-10 and induces lymphoma cell apoptosis [63].

IL-10 and cancer

Role of IL-10 in cancer though well accepted is vaguely understood. IL-10 is known to exhibit both pro and anti-tumor activities. IL-10 exhibits tumor regression activity [64]. Some proposed that IL-10 antitumor effect is due to enhanced NK cell activity [64], while others have demonstrated that anti-tumor effect depends on CD8+ or CD4+ T cell function [65]. But, in contrast several studies proposed that IL-10 may reduce immune response against cancer [34, 66]. IL-10 possibly acts as a negative mediator in the cross-talk between innate and adaptive antitumor immunity. The other mechanism of immunosuppression of anti-cancer immunity is thought to be mediated via tumor-derived factors inducing DC dysfunction and particularly alteration of DC differentiation, maturation and longevity as a mechanism for immune suppression. IL-10 is potent inhibitor of DC and thus possibly involved in reduction of anti-cancer immunity [66]. Recently, Mocellin et al. [65] reviewed both aspects of IL-10 in cancer immunity.

Cancer is illustrated by few common manifestations like, evasion of apoptosis, insensitivity to growth signals, induction of angiogenesis as proposed by Hanahan and Weinberg [67]. Various studies have reported effect of IL-10 on most of these hallmarks of tumorogenesis. IL-10 suppresses peripheral blood T cell apoptosis in vitro by increasing Bcl-2 expression [68]. IL-10 also prevents T cell apoptosis on IL-2 withdrawal and Epstein-Barr virus infections through upregulation of Bcl-2 expression [69]. Bcl-2 overexpression is attributable to activation of signal transducer and activator of transcription 3 (STAT 3) by IL-10 through autocrine or paracrine loops in lymphoma cells [70]. IL-10 could also promote Bax mRNA expression in culture-activated hepatic stellate cells [71]. Recently, IL-10 has been implicated in resistance to apoptosis in lung cancer [72]. p53 is an important tumor suppressor having inhibitory activity on IL-10 [73]. High IL-10 production is also observed in lymphocytes from p53-deficient mice with experimental autoimmune encephalomyelitis [74]. Cytokine promoters can be repressed by p53, acting as a negative regulator of these cytokines [75]. Contradictory results have been observed in relation to role of IL-10 in angiogenesis in tumor. IL-10 has been reported to exert anti-angiogenic activity in several cancers [76, 77]. Whereas some other report suggest that IL-10 may promote angiogenesis [78, 79].

IL-10 as breast tumor inhibiting cytokine

Although tumor promoting activities of IL-10 are known, it is predominantly reported to have anti-tumor property. Some of the proposed mechanisms of anti-cancer activity of IL-10 includes- activation of NK-cells [64, 79], synergistic activation of cytotoxic T lymphocyte for maintenance of CD8+ [80] and CD4+ mediated [81] anti-tumor response, enhancement in surface expression of MHC antigen for maintaining susceptibility of cancer cells to NK-cells [82], enhancement in tumor infiltration by neutrophil and macrophages, and finally modulation of angiogenesis and invasiveness through inhibition of metalloproteinase [77, 79, 83, 84].

Kundu et al. [64] studied anti-tumor and anti-metastatic properties of IL-10 in murine model and observed that tumorigenicity in immunocompetent mice was significantly abrogated by IL-10. Later, it was found that mice subjected to immunization with IL-10 expressing tumor cells promoted the loss of tumorigenicity and induced a protective anti-tumor immune response which was mediated either by NK cells or CD8+ T cells [85]. Table 1 summarizes various studies with IL-10 which describes its tumor inhibiting action particularly in breast cancer.

Table 1 Effect of IL-10 in breast cancer cells and tumor
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IL-10 as breast tumor promoting cytokine

IL-10, a well-established suppressor of immunity reduces the antigen presentation capacity of macrophages and inhibits production of several cytokines which have important role in tumor immunosurveillance. Therefore, higher IL-10 level may facilitate tumor immune escape. This observation has been reported in various types of cancers and is supported by in vivo IL-10 knock out studies. IL-10 knockout mice show increased survival and more bladder tumor rejection compared to normal mice [90] indicating inhibitory effect of endogenous IL-10 on tumor immunosurveillance system, thus, help in tumor initiation and growth. But, only few studies in breast cancer cell line or patient are available that supports direct pro-tumor action of IL-10 in breast cancer. These studies reporting breast tumor promoting activities of IL-10 are highlighted in Table 1.

IL-10 as prognostic indicator for breast cancer

Tumor progression is determined by intricate interaction of tumor cells, stromal cells and T lymphocytes. IL-10 produced by tumor cells and immune cells play an important role in tumor cell growth and proliferation in tumor microenvironment. Increased IL-10 concentration is frequently detected in serum of breast cancer patients. It is proposed that IL-10 is secreted at a higher rate by metastatic cancer cells for down-regulating inflammatory response of cell-mediated immunity [91]. IL-6 is known to promote tumor growth by up regulating anti-apoptotic and angiogenic protein in tumor cells [92–94] and elevated IL-10 may inhibit tumor growth by suppressing IL-6 production. This is supported by observation of inverse correlation of IL-10 and IL-6 levels in breast cancer patients [95]. But, IL-6 level reported both as positive and negative prognosticator of breast cancer [92]. Therefore, such inverse correlations with IL-6 level may raise question on any definitive role of IL-10 in breast cancer prognosis.

IL-10 is over expressed in estrogen receptor (ER)-negative breast tumor in comparison to ER-positive tumors [96]. Breast cancer patients with prolactin receptor (PR) positive tumor have lower IL-10 level. AP-1 expression is higher in ER-negative tumors than ER-positive tumors and higher AP-1 expression correlate with high IL-10 level [96].

Significant differences in IL-10 cytokine level reported in breast cancer patients and negative breast biopsy group. IL-10 showed more than 50% difference among cancer and negative biopsy group [97].

Akbulut et al. [98] in a tumor model generated with, NT-2 cell (Neu transgenic spontaneous mouse mammary tumor-derived cell line) found that IL-10 level is higher in serum than tumor tissue and reduced by vaccination with tumor associated antigen (TAA) vector vaccine as well as chemotherapy.

Kozlowski et al. [95] found that serum IL-10 is strongly associated with breast cancer. In their study with 45 breast cancer patients and 25 normal subjects, mean IL-10 level in control samples was 5.7 pg/ml in comparison to 24.7 pg/ml in patient samples. Serum level of IL-10 was elevated in 35 (77.8%) patients. However, serum IL-10 level was not associated with any of the stages of breast cancer patient grouped using TNM classification [95]. Another study in 90 breast cancer patients and 15 healthy volunteers found no differences in baseline IL-10 levels between cancer patients and healthy volunteers [99]. However, 45% of patients had measurable plasma IL-10 level and weekly paclitaxal treatment caused transient increase. But other report indicates that paclitaxel treatment significantly decreases IL-10 levels [100]. A recent study have also reported lack of significant correlation of IL-10 level with either pathologic or clinical response in locally advanced breast cancer patients treated with neoadjuvant chemotherapy [101].

Llanes-Fernándeza et al. [102] reported that 85% (23 out of 27) of breast cancer tissue examined showed strong IL-10 expression. IL-10 was significantly associated with apoptosis markers. An inverse association of IL-10 with p53 and a positive association between IL-10, Bcl-2, and Bax were observed. 89% of patients negative for p53 were found to express IL-10. Presence of IL-10 and higher expression of Bcl-2 family proteins in tumor microenvironment are proposed to represent an increase in breast tumor aggressiveness [102].

Merendino et al. [103] reported correlation between IL-10 levels and clinical stages of breast cancer. Neoplastic metastatic disease was found to be associated with higher IL-10 levels (1002.8 ± 425.7 pg/ml, N = 10) compared with patients with non-metastatic disease (238.3 ± 103.8 pg/ml, N = 10). IL-10 level of both groups of cancer patients were also significantly higher (P < 0.05) than those of healthy donor (7.6 ± 5.7 pg/ml, N = 10). It was proposed that presence of IL-10 in sera of cancer-bearing patients possibly contributes in decreasing immune surveillance favoring tumor development. It has been suggested that IL-10 suppresses Th-1 cell development through down-regulation of IL-12 production by monocytes and favor generation of Th-2 developmental pathways, inducing humoral response and immunosuppression of cell-mediated immunity [103]. In a study conducted later by the same group [104], reported that mononuclear cells obtained from breast cancer patients exhibit a defective IL-12 production capability and generate higher amounts of IL-10.

Son et al. [105] in a recent study investigated methylation status of IL-10 gene in 30 normal, 31 benign, and 72 breast cancer paraffin-embedded tissue specimens from the National Cancer Center, Korea. Significantly lower methylation rates of the IL-10 gene in malignant tumors were observed than that of benign and normal tissues. Tissues with aberrant methylation of IL-10 gene showed significantly lower rates of mRNA expression. Whereas unmethylated IL-10 showed approximately 10,000-fold upregulated mRNA expression compared to those with IL-10 methylation. IL-10 methylation also showed significant association with lower Ki-67 expression. It was proposed that hypomethylation influences IL-10 gene activation and the process of breast carcinogenesis [105].

In another recent study [106] on adipose derived stem cells (ASC) isolated from breast cancer patients, significantly high mRNA expressions of IL-10 and TGF-β1 were reported than those from normal individuals. The culture supernatant of ASCs induced upregulation of mRNA expression levels of IL-4, TGF-β1, IL-10, CCR4, and CD25 in peripheral blood leukocytes. When the same culture supernatant was added to ASCs isolated from normal subjects augmentation of mRNA expressions of IL-4, IL-10, IL-8, MMP2, VEGF, and SDF-1 in normal ASCs was also observed [106]. This report for the first time suggest that resident derived stem cells in breast cancer tissue have crucial roles in inducing regulatory molecules like IL-10 and thereby promoting anti-inflammatory reaction within the tumor microenvironment favorable for tumor growth.

IL-10 transcripts were also found to be expressed in 14/15 primary breast adenocarcinomas and in 5/8 established breast tumor lines [107]. This supported by immunohistochemistry and immunoprecipitation from lysates and supernatants showed that established breast tumor lines produces IL-10 protein. IL-10 is localized not only in tumor cells of primary breast adenocarcinomas but also in occasional infiltrating mononuclear cells.

These findings of increased serum and transcript level of IL-10 and altered methylation pattern in breast cancer suggest that it may have utility in describing cancer prognosis along with other existing panel of molecular prognosticator. However, more elaborate multicentre-validated studies will be required before any such consideration. Various studies on expression and level of IL-10 in breast cancer and their significance in relation to breast cancer are summarized in Table 2.

Table 2 Expression of IL-10 in breast cancer patients
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IL-10 as genetic biomarker of disease risk

Genetic polymorphic variants have been recently investigated as genetic marker of possible risk for initiation and progression of common and complex diseases like breast cancer. Several candidate genes have been identified which may correlate with risk of breast cancer and its progressive stages. IL-10 contains several well-characterized polymorphic sites which have been screened in breast cancer patients to find if any of these are involved with breast cancer risk. IL-10-1082 (A>G); -819 (C>T); -592 (C>A) SNPs are most commonly studied in breast cancer patients. Majority of IL-10 polymorphism studies observed significant association of IL-10 with breast cancer with exception of one study [110] and a recent meta-analysis [111] which could not find association of IL-10 with breast cancer. Genetic polymorphism studies on IL-10 in breast cancer conducted till today are summarized in Table 3.

Table 3 Genetic evidences of nature of association of IL-10 polymorphism with breast cancer
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Although polymorphism studies are influenced by several factors like ethnicity of study population, sample size and inclusion, exclusion criteria, etc., studies indicating association of IL-10 with breast cancer may further confirm the role of IL-10 in breast cancer. IL-10 gene promoter polymorphism is already known to influence IL-10 production [117–119]. In breast cancer patient also, enhanced IL-10 level and correlation with advancing stages were reported. However, studies with larger population size of patients from different racial and ethnic origin will be required for deriving definitive conclusions. Besides, stringent application of in silico analysis for genome-wide SNP interactions of combinations of genes would be required to address current limits of polymorphism studies in breast cancer [120].

In summary, growing evidences suggest that IL-10 has important role in initiation and progression of breast cancer, although much of the intricate mechanism involved are not thoroughly investigated. IL-10 predominantly exerts tumor inhibiting action on breast cancer, however, it also has potential to promote tumor. This dual nature of IL-10 may further be dependent on temporo-spatial expression of IL-10 and level available. Majority of genetic studies points toward a significant correlation of IL-10 and breast cancer. This is also supported by elevated serum IL-10 level in breast cancer patients. Due to its complex dual role in tumor initiation and progression, any attempt to manage tumor with IL-10 as therapy would not be that simple and straight forward. However, IL-10 may serve as a crucial biomarker with certain amount of prognostic significance. Further studies on IL-10 are expected to shed more light on these contentious areas so as to convincingly establish its true nature of association with breast cancer.