As IMiDs, thalidomide and its analogues have clinically relevant effects on cytokine elaboration (5). In patients with ENL, a Mycobacterial infection complication, treatment with thalidomide reduced the lipopolysaccharide (LPS)-induced TNF-α secretion by monocytes or macrophages through accelerating TNF-α mRNA degradation and inhibiting nuclear factor κB (NF-κB) activation (Fig. 1) (8–10). Thalidomide has also been demonstrated to exert an effect on other inflammatory conditions that present with TNF-α upregulation, such as rheumatoid arthritis, Crohn's disease, tuberculosis and cancer (11–14). Furthermore, in the setting of spinal cord ischemia, thalidomide pretreatment has been demonstrated to reduce early phase ischemia/reperfusion injury of the spinal cord in rabbits via reducing TNF-α production (15). CC5013 and CC4017 are considered to be more effective TNF-α antagonists than thalidomide, and their inhibiting effect on TNF-α production has been reported as being as much as 100–50,000 times higher (16,17).

The TGF-β superfamily consists of various proteins that regulate numerous physiological processes, including embryogenesis, immunity, carcinogenesis, cell proliferation and migration, wound healing, inflammation and fibrosis, among others (18). Smad and non-canonical pathways are the two primary signaling pathways in the TGF-β system (18). In Smad-related pathways, complex interactions between intracellular diverse molecules associated with the downstream TGF-β signaling cascade result in fibrosis, such as phosphorylation or direct interaction with Smad (19). Non-canonical signaling pathways include the mitogen-activated protein kinase (MAPK), Rho-like GTPase and phosphatidylinositol-3-kinase/protein kinase B (AKT) pathways (18). Extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun-NH₂-terminal kinase, and p38 MAPK signaling pathways are well-established non-canonical pathways of TGF-β signaling (20). In a mouse model of bleomycin-induced lung fibrosis, Choe et al (21) demonstrated that thalidomide administration significantly inhibited TGF-β1 mRNA expression in a dose-dependent manner following the administration of interleukin (IL)-6 and IL-6R, and they concluded that the anti-fibrotic effects of thalidomide may be associated with the inhibition of TGF-β1-mediated non-Smad ERK1/2 signaling pathways. Furthermore, Liang et al (22) found that thalidomide treatment inhibited fibronectin production in normal and keloid fibroblasts stimulated by TGF-β1, and enhanced the phosphorylation of MAPKs and Smad 2/3. They also revealed that inhibited p38/Smad3 pathways were involved in the anti-fibrotic mechanisms of thalidomide (Fig. 1) (22). Other than TNF-α and TGF-β, the expression levels of other cytokines including IL-1β, IL-6, and granulocyte macrophage-colony stimulating factor were also suppressed by thalidomide in LPS-induced peripheral blood monocytes, whereas IL-10 was stimulated (23,24).

Li et al (25) demonstrated that the derivatives of thalidomide were able to downregulate CCAAT-/enhancer-binding protein-β (C/EBP-β)-related cell proliferation and inhibit 5′-cap-dependent translation of C/EBP, both of which are associated with eukaryotic translation initiation factor 4E (eIF4E) (25). Cyclooxygenase (COX)-2, a key regulator in arachidonic acid metabolism and prostaglandin generation, is an important anti-inflammatory target (26). LPS-induced COX-2 expression is transcriptionally regulated by C/EBP isoforms (27); therefore, IMiDs may be directly involved in the eIF4E-dependent regulation of COX-2 expression. Furthermore, selective upregulation of COX-2 expression has been observed in myeloma cells, and COX-2 inhibition in these cells may eliminate the protective effects of bone marrow stromal cells on myeloma following chemotherapy (28). A phase II clinical trial found that selective inhibition of COX-2 by thalidomide has an effect in multiple myeloma (MM) (29), which suggests that the anti-inflammatory effect of IMiDs may be relevant to COX-2 inhibition.

Correlative preclinical studies have suggested that IMiDs act as co-stimulators on the release of IL-2 and interferon (IFN)-γ from T lymphocytes undergoing CD3 ligation or mitogenic stimulation, or in response to dendritic cells presented with antigens (30). This may result in T cell proliferation, T helper cells skewing and augmentation of cytotoxic effector functions (31). Co-stimulation of T cells by lenalidomide is only partially blocked by the negative co-stimulator cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in the B7/CD28 co-stimulatory axis (32). Furthermore, tyrosine phosphorylation of CD28 induced by lenalidomide was able to facilitate the recruitment and activation of upstream molecules in multiple intracellular signaling pathways, including phosphatidylinositol-3-kinase (PI3K) and NF-κB (33). As CD28 and IMiDs have no kinase activity, other signaling proteins such as protein kinase C (PKC) may be involved in the indirect mechanism underlying CD28 phosphorylation (34,35). Payvandi et al (35) demonstrated that IMiDs increased IL-2 production in stimulated T cells via enhancing PKC-θ activation and DNA-binding activity of activated protein-1 (AP-1). In addition to conventional T cells, regulatory T cells (Tregs) are a group of immunosuppressive T cells that function in self-tolerance and the immune response (36). Inhibition of Tregs by lenalidomide (Revlimid; CC-5013) and pomalidomide (CC-4047) via decreasing the expression of forkhead box P3 (FoxP3) was observed in a preclinical study (32). Furthermore, Gandhi et al (37) reported that the lenalidomide (or pomalidomide)-binding protein cereblon was able to cause the Ikaros and Aiolos to interact with the E3 ubiquitin ligase CUL4-RBX1-DDB1-CRBN (known as CRL4CRBN), resulting in their ubiquitination, proteasome degradation and T cell activation.

IMiDs also exert their immunomodulatory effects by modulating the number and function of NK cells. Zhu et al (38) reported that CC-5013 and CC-4047 increased the population of NK and NKT cells in the setting of hematological malignancy (38). Furthermore, Wu et al (39) demonstrated that lenalidomide was able to enhance the NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) effect on rituximab-treated CD20+ tumor cells (39). It was also revealed that enhanced NK cell Fc-gamma receptor signaling was associated with enhanced phosphorylation of ERK (39).

Neovascularization, a well-known consequence of the inflammatory response (31), provides an insight into the inherent connection between inflammatory cytokines and angiogenic pathway. Yaccoby et al (40) demonstrated that peritoneal injection with thalidomide inhibited myeloma growth in severe combined immunodeficiency-human (SCID-hu) mice implanted with fetal human liver fragments. Reduced micro vessel density was observed in myeloma xenografts of thalidomide-treated fetal liver-bearing mice compared with non-fetal liver implanted hosts. They concluded that thalidomide treatment is strongly correlated with decreased micro vessel density; however, they did not determine whether the reduction in microvascular density is one of the main results of thalidomide antiangiogenic activity or if it was a secondary process to reduce tumor burden. In a rat model of hepatic cirrhosis, Lv et al (41) reported that thalidomide was able to downregulate NF-κB-induced expression of adhesion molecules (such as intercellular adhesion molecule-1) and activation of hepatic stellate cells via inhibiting the degradation of IκB to reverse the disease. In the setting of hereditary hemorrhagic telangiectasia, thalidomide treatment stimulated mural cell coverage and restored vessel wall defects via increasing the platelet-derived growth factor-B expression in endothelial cells and stimulating mural cell activation (42).

Despite the putative role of metabolism in the IMiD-mediated inhibition of vascularization, thalidomide and its analogues have been demonstrated to have an antagonistic effect on several key angiogenesis regulators (31). IMiDs decrease the paracrine production of pro-angiogenic vascular endothelial growth factor (VEGF) and IL-6 at the bone marrow stroma and MM cell levels (43). Furthermore, thalidomide suppresses the expression of VEGF receptors in human umbilical vein endothelial cells (HUVECs) via activating sphingolipid signaling (44). Reduced cell migration responses to basic fibroblast growth factor (bFGF) and VEGF due to downregulation of AKT phosphorylation was observed in lenalidomide treated HUVEC cells (45,46). The suppression of pro-angiogenic factors such as IL-6, TNF-α, VEGF, NF-κB and prostaglandin synthesis by thalidomide and its analogues may indirectly downregulate angiogenesis in vivo.

Skin fibrosis, characterized by physiological tissue architecture damage, is a major hallmark of systemic sclerosis (SSc) and causes high morbidity among patients (47). As efficient immunomodulators, thalidomide and its analogues have been suggested as a treatment for treat skin fibrosis. Oliver et al (48) reported that thalidomide improved the clinical manifestations of patients with SSc by stimulating the immune response. In a preclinical study by Weingärtner et al (49), the newest second generation IMiD, pomalidomide, was demonstrated to prevent spontaneous hypodermal fibrosis and induce its regression in bleomycin-induced dermal fibrosis mice and tight-skin mice. An international, multicenter, controlled phase II clinical trial (gov ID: NCT01559129) was initiated to evaluate pomalidomide as a treatment for patients with SSc (49). Most recently, Ingen-Housz-Oro et al (50) reported that two cases of Ig4-related skin diseases were successfully treated with thalidomide, and they concluded that interactions between Tregs and mast cells may result in IL-6 production and fibrosis via the TGF-β/phospho-Smad 2/3 pathway.

Idiopathic pulmonary fibrosis (IPF), characterized by epithelial injury and fibroblast proliferation in the lungs, is a chronic progressive lung disease with a prevalence of 16–18 in 10,000 and a five-year mortality rate over 50% (51). Inflammatory cytokines and angiogenesis serve critical roles in the pathological progression of IPF. It has been reported that thalidomide exerts an inhibitory effect on the cytokine profile in patients with IPF (52,53). Tabata et al (54) reported that thalidomide pretreatment prevented the development of bleomycin-induced pulmonary fibrosis (PF) in a mouse model. Choe et al (21) further confirmed that the TGF-β1-induced activation of the ERK1/2 signaling pathway accounted for the anti-fibrotic effect of thalidomide in a bleomycin-induced PF model. Knobloch et al (55) reported that the underlying mechanisms of this effect included downregulation of bone morphogenetic protein signaling, an increase in the activity of Wnt and Akt and enhanced apoptosis resistance. In a clinical trial conducted by Horton et al (56), thalidomide treatment was demonstrated to improve coughing and respiratory symptoms, as well as quality of life, in patients with IPF.

D'Amato, an ophthalmologist, first considered thalidomide when searching for treatments for two diseases that cause blindness, macular degeneration and diabetic retinopathy (57). Both diseases result in excessive blood vessel growth, which burst in the eye and destroy the patient's vision (57). It was reported that thalidomide was an effective anti-angiogenesis inhibitor in the setting of bFGF-induced angiogenesis in a rabbit cornea micropocket assay (58), and Kruse et al (59) suggested that thalidomide inhibited VEGF-induced corneal angiogenesis in New Zealand white rabbits. Ribeiro et al (60) used a novel thalidomide hybrid (LASSBio-596) to treat inflammatory corneal angiogenesis in rabbits, and reported that it had an inhibitory effect. In an alkali burn model of corneal angiogenesis, Abbas et al (61) demonstrated that thalidomide was able to prevent corneal angiogenesis and prolong graft survival (as measured by graft clarity in donor corneas in eyes). Furthermore, Lee and Chung (62) has reported a similar inhibitory effect of thalidomide on corneal neovascularization in a silk suture-induced rabbit model, though to be achieved via regulating VEGF and TNF-α expression levels. However, Srinivasan et al (63) reported the case of a patient who presented with bilateral symmetrical corneal endothelial changes, which was found to be associated with thalidomide toxicity. Huang et al (64) reported a 45-year woman with gradual visual decline due to crystalline materials in the cornea. The patient was treated with thalidomide and, at a seven-year follow up, her vision was stable and the corneal crystalline deposits had decreased and migrated.

In addition to functioning as an anti-angiogenesis inhibitor, thalidomide may also used as anti-inflammatory agent in ophthalmopathy. In a rat model of endotoxin-induced uveitis (EIU), Guex-Crosier et al (65) revealed that high-dose thalidomide had an effective anti-inflammatory effect, whereas lower doses were insufficient to reduce inflammation. Rodrigues et al (66) further investigated the preventive and therapeutic anti-inflammatory effects of systemic and topical thalidomide in EIU rats. Parentin et al (67) reported that thalidomide was an effective treatment for bilateral chronic idiopathic anterior uveitis in a three-year-old child; however, Ip et al (68) reported a case in which thalidomide was not able to prevent the recurrence of a choroidal neovascular membrane (CNM). Using a mouse model, Rabinowitz et al (69) also demonstrated that thalidomide had no significant effect on neovascularization in oxygen-induced retinopathy.

It has been reported that exposure to thalidomide has negative side effects on the eyes. For example, anophthalmia and microphthalmia (the absence of an eye and the presence of a small eye within the orbit, respectively) are both disease caused by exposure to thalidomide (70). There have been no large sample clinical trials investigating the effect of thalidomide on ophthalmopathies, and so the application of thalidomide in the patients with certain types of eye diseases remains controversial.