Immunosuppressive tumor microenvironment and uterine fibroids: Role in collagen synthesis.

Despite the high prevalence of uterine fibroids and their health and economic consequences, little research has discussed the possible immunological causes of the disease. Immune reprogramming is one of the possible causes of UF formation through inducing collagen synthesis. The increase in M2 macrophage, MDSCs, and TH17 are among the most possible causes for inducing collagen synthesis and thus UF formation. This review represents a new era towards more investigations of the change in the UF immune microenvironment and also possible targeting approaches, through immunomodulation, that leads to better prognosis of UF cases.

Uterine fibroids (UF), also known as leiomyoma, is a benign uterine tumor with an unclear etiology that predominately affects the myometrium. Most of these monoclonal, estrogen-dependent uterine neoformations affect women in their reproductive years and 80% of them experience symptoms for the rest of their lives. Uterine fibroids can cause anemia from prolonged or heavy monthly flow, pelvic pain, or pressure, and difficulties in achieving successful pregnancies. UF develops as a result of changes of myometrial microenvironment under particular pathological circumstances [ 1 ]. It is unclear exactly what molecular and cellular alterations caused the UF to form and grow. UF are distinguished by an excessive buildup of extracellular matrix (ECM) elements like collagen, fibronectin, laminins, and proteoglycans. ECM deposition is thought to be the root of aberrant bleeding and pelvic pressure or pain [ 2 ]. ECM proteins induce mechanotransduction, a mechanism wherein increased tissue stiffness leads to bidirectional signaling via integrins and downstream mediators like Rho/p38 MAPK/ERK [ 3 ]. To control these tumors, it may be possible to prevent additional ECM formation and the associated fibrosis [ 4 ]. Additionally, uterine fibroids express proteolytic enzymes that are essential for ECM remodeling, such as matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). MMPs and other proteolytic enzymes release growth factors and activate numerous signal transduction pathways by destroying ECM components. Growth factors and steroid hormones control how much ECM accumulates and how it functions. Growth factors are bound and hidden by the ECM, which increases their stability and limits their activity. Among the suggested predisposing factors for ECM deposition is the shift in immune microenvironment from the proinflammatory type 1 immunity to the anti-inflammatory type 2 immunity [ 5 ]. Type 2 immunity is characterized, to some extent, as a counter-regulatory process controlling type 1 immunity and exhibits both host-protective and pathogenic qualities. Type 2 immunity promotes resolution of the inflammatory process after an injury. Chronic stimulation of type 2 response leads to tissue fibrosis [ 6 ]. The cytokines interleukin 4 (IL-4), IL-5, IL-9, and IL-13 are produced more frequently in type 2 immunity [ 7 ]. In this review, we highlight the involvement of type 2 immunosuppressive cells such as M2 macrophage, MDSCs, and Th17 in UF pathogenesis by affecting ECM accumulation Macrophages are myeloid immune cells that exhibit a propensity for enthusiastic phagocytosis [ 8 ]. Macrophages serve a crucial part in the body’s homeostatic maintenance by eliminating internal waste and repairing tissue, in addition to acting as a defense against infections like bacteria. However, because macrophages can act as effectors for cell-mediated immunity and present antigens to T cells, it is recognized that they play a role in the onset of infectious illnesses, malignancies, and chronic inflammatory conditions including arteriosclerosis [ 9 ]. Macrophages can develop a variety of states depending on their environment, which are roughly categorized into two main groups: M1-like or pro-inflammatory and M2-like or anti-inflammatory [ 10 ]. Interferon-gamma (IFN-γ), TNF, granulocyte-macrophage colony-stimulating factor (GM-CSF), and various pathogen-derived signals, such as lipopolysaccharide (LPS), induce the formation of M1-like macrophages. M1 macrophages entrap, phagocytose, and lyse tumor cells. Additionally, M1’s have the capacity to deliver tumor antigens which would encourage other leukocytes to perform cytotoxic actions [ 11 ]. For instance, immunostimulatory cytokines (IL-6, IL-12, and tumor necrosis factor (TNF)) produced by M1 phenotype macrophages may boost CD8 + T cells and NK cells, initiating apoptosis in tumor cells [ 12 ]. M1 phenotype secrete high levels of pro-inflammatory molecules like TNF, IL-1, and IL-12, carry out efficient antigen presentation and co-stimulation, and regulate defense mechanisms against infections and cancer [ 13 ]. Less immunogenic antigens are present in tumor stem cells, but these cells are more active in proliferating and differentiating. Given the heterogeneity and tumor-specific immune escape mechanisms, tumor stem cells may use M1 phenotype macrophages as a natural filter to evade killing and advance to the next stage. With low antigenicity, most macrophages transit from the M1 phenotype to the M2 phenotype, which is more likely to contain immunosuppressive potentials in tumor nests [ 14 ]. In addition to the pathogen defense, M2 macrophages clear apoptotic cells, decrease inflammatory responses, and induce wound healing. M2 phenotype macrophages support tumor growth by generating growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β1), hepatocyte growth factor (HGF), and b-fibroblast growth factor (b-FGF) [ 15 ]. In actuality, all macrophages are important for the growth of the initial tumor nest and encourage subsequent spread of the tumor. In light of M1/M2 ratios and responses, immunoediting theory suggests that tumor growth can be viewed as stages of immunological eradication, equilibrium, and escape [ 16 ]. M2 macrophage was found to induce collagen synthesis and thus upregulation of UFs through secretion of IL-4, IL-10, IL-13, and TGF-β1 ( Figure 2 ). In addition to Th2 lymphocyte; basophils, mast cells, and eosinophils are the main producers of IL-4. La Flamme, Kharkrang [ 17 ] results supported that type II macrophages (M2 macrophages) as an important innate immune source of IL-4. The role of the IL-4 protein is to induce the development of naive helper T lymphocyte cells into T-helper-2 cells (TH2), as well as the production of several other cytokines, such as IL-13, IL-5, and IL-10 [ 18 ]. Sosna, Kolesár [ 19 ] found an association between polymorphisms of the IL4 gene promotor at positions −590 C/T and −33 C/T and observed the risk of leiomyoma. Interleukin 4 is expected to induce UF collagen synthesis through multiple effects. Compared to myometrium progenitor cells, leiomyoma progenitor cells exhibited significantly (p < 0.05) higher levels of IL4 [ 20 ]. Krsteski, Jurgec [ 21 ] showed that rs1801275 (IL4R) can predispose patients to develop multiple UF. Măluţan, Drugan [ 22 ] found that women with endometriosis had considerably higher mean serum levels of IL-1Ra, IL-4, and IL-10 than did women in the control group without the condition. Aoudjehane, Pissaia [ 23 ] found that IL-4 has profibrotic effects by activating intrahepatic myofibroblasts and inducing collagen synthesis and release which requires STAT-6 activation and IL4-R binding. Liang, Zhang [ 24 ] showed that IL-4 stimulated the synthesis of fibronectin and α-smooth muscle actin (α-SMA), and STAT6 in mouse bone marrow monocytes. Knipper, Willenborg [ 25 ] showed that lysyl hydroxylase 2 (LH2), an enzyme that controls enduring pro-fibrotic collagen cross-links, is induced in fibroblasts by IL-4R signaling in macrophages. Sugimoto, Enjoji [ 26 ] showed that in comparison to untreated cells, treatment of LI90 cells with IL-4 enhanced collagen I synthesis. In a JAK/STAT-dependent way, IL-4 strongly and selectively stimulates prostate stromal fibroblasts to produce collagen mRNA and protein [ 27 ]. Interleukin-10 (IL-10) is a secreted inhibitory cytokine that is produced by T helper (Th) 2 cells and M2 macrophages and has been shown to prevent Th1 cells from producing cytokines [ 28 ]. M2 macrophages produce more IL-10 and other anti-inflammatory cytokines than other macrophage subtypes [ 29 ]. Antigen-presenting cells (APCs), which include dendritic cells (DCs), Langerhans cells, and macrophages, are just a few examples of how IL-10 affects both innate and adaptive immunity [ 30 ]. IL-10 inhibits the antigen-presenting ability of monocytes by down-regulating class II MHC, hence preventing the development of human T lymphocytes that are specific for antigen [ 31 ]. Compared to myometrium progenitor cells, leiomyoma progenitor cells exhibited a significant 40% increase in IL10 levels and significantly lower levels of Th1/Th17 cytokines (IL6, IL12, IL17A, IFN-γ, G-CSF, and TGF-β1) [ 20 ]. Jung, Ma [ 32 ] found that in vivo IL-10 administration induced an increase in fibroblast activation (proliferation, migration, and collagen synthesis). Matsuzaki, Pouly [ 33 ] showed that endometriotic stromal cells developed a pro-fibrotic phenotype as a result of IL-10, including increased cell division, the production of collagen type I, the appearance of positive stress fibers for smooth muscle actin, and contraction of collagen gel. Chang, Liu [ 34 ] showed that with the development of endometriosis, IL-10 + Th17 cells in the endometriotic milieu rapidly increased. Yang, Zhou [ 35 ] results suggest that by increasing the release of IL-10 and TGF-β, the interaction between macrophages and endometrial stromal cells may reduce the cytotoxicity of NK cells. It may also cause immunological escape of ectopic pieces and encourage the onset and progression of endometriosis. Matsuzaki, Pouly [ 33 ] found that endometriotic stromal cells underwent pro-fibrotic phenotypic induction by IL-10, including cell proliferation, collagen type I synthesis, α-SMA positive stress fibers, and collagen gel contraction which was reduced by STAT3 gene knockdown. On the other hand, IL-10 showed no appreciable influence on the pro-fibrotic phenotype of healthy women’s endometrial stromal cells. Bhattacharyya, Boostanpour [ 36 ] findings reveal a novel, IL10-dependent mechanism of macrophage polarization leading to fibroblast activation after injury. A crucial T helper 2 (Th2) cell-derived cytokine called IL-13 has been shown to play a role in regulating both pathological processes like autoimmune diseases, bronchial asthma, and organ fibrosis as well as normal physiological processes like inflammation, immune response, mucus production, and tissue reconstruction [ 37 ]. Li, Liu [ 38 ] showed that IL-13 increased expression of CCR4, CCL19, CCL21, and CXCL2 which induces Treg activation and recruitment, supporting its immunosuppressive role. Krsteski, Jurgec [ 21 ] found that rs20541 (IL-13) may contribute to susceptibility of UF development. Compared to myometrium progenitor cells, leiomyoma progenitor cells exhibited significantly (p < 0.05) higher levels of IL13 [ 20 ]. Firszt, Francisco [ 39 ] found that when compared to healthy controls, IL-13, dramatically increased the formation of collagen type-1 in asthma patients and thus airway remodeling Through a mechanism involving TGF-β1 and MMP-2, IL-13 enhances airway remodeling. O’Reilly, Ciechomska [ 40 ] showed that IL-13 increased collagen expression in a STAT6-dependent way. Sugimoto, Enjoji [ 41 ] results suggested that HSCs were directly impacted by IL-13, which increased collagen synthesis and decreased cell division. Data from Bailey, Bland [ 42 ] indicate that the IL-13 pathway is activated in the fibrotic intestinal muscle of Crohn’s patients and it inhibits fibroblast MMP synthesis and down-regulates matrix degradation, resulting in excessive collagen deposition. Lu, Liu [ 43 ] demonstrated that in primary cultured mouse airway fibroblasts, IL-13 dose- and time-dependently increased collagen synthesis which was countered by inhibiting the JAK/STAT6 signal pathway. Cardilo-Reis, Gruber [ 44 ] showed that IL-13 administration promotes collagen production in established lesions of atherosclerosis-prone mice. Transforming growth factor-beta1 (TGF-β) is a cytokine that regulates many cell-intrinsic processes that reprogram the tumor microenvironment. The basic processes of cell and tissue development, vasculogenesis, wound healing, and immunological homeostasis are all controlled by TGF-β1 [ 45 ]. TGF-β acts as an immunosuppressive cytokine that induces growth, differentiation, tolerance induction, and homeostasis of immune cells [ 46 , 47 ]. Increased amounts of TGF-β and other anti-inflammatory cytokines are expressed by M2 macrophages. In fibroblast cocultured UtLM, an increased secretion of TGF-β1,3 was found, and immunoprecipitated TGF-β receptor I from cocultured UtLM cells showed elevated phosphoserine expression [ 48 ]. Findings from Hillege, Galli Caro [ 49 ] revealed that TGF-β acutely enhanced the expression of a subset of fibrosis-related genes, including CTGF and FGF-2, in both myoblasts and myotubes, which was then followed by an increase in Col1a1 expression. Pan, Chen [ 50 ] results showed that in cardiac fibroblasts, TGF-β1 increased COL1A1 mRNA expression and protein synthesis. When cells were exposed to 10 ng/mL of TGF-β1 for 48 hours, the expression of DNA methyltransferase 1 (DNMT1) and DNMT3a was considerably downregulated, and the overall DNMT activity was suppressed. The rat COL1A1 promoter’s DNA methylation percentage was significantly reduced after TGF-β1 treatment at numerous CpG sites and thus inhibiting DNMT1 and DNMT3a expressions as well as overall DNMT activity and subsequently increase collagen type I expression. Myeloid-derived suppressor cells (MDSCs) are heterogeneous populations of immature myeloid cells that exhibit the capacity to inhibit both innate and adaptive immune responses. They are divided into two subtypes, polymorphonuclear (PMN-MDSCs) or granulocytic MDSCs, which morphologically and phenotypically resemble neutrophils, and monocytic MDSCs (M-MDSCs), which morphologically and phenotypically resemble monocytes [ 51 ]. Clinical research found that patients with solid tumors who had circulating and/or infiltrating MDSCs at the tumor site had a poor prognosis. MDSC removal could aid in reestablishing immunological surveillance [ 52 ]. Kim, Chu [ 53 ] showed that expression of programmed death ligand 1 increases MDSCs accumulation, promotes tumor progression by gastric epithelial cells. Lebrun, Lo Re [ 54 ] data showed that silica-treated mice’s M-MDSCs produced TGF-β1 and that resulted in which increased TIMP-1 release by lung fibroblasts. This inhibited collagen breakdown and facilitated lung fibrosis. In addition to their immunosuppressive effects, sB7H3-mediated disease progression may be influenced by TERT and B7H3-dependent MDSC recruitment/expansion from BM, which may trigger myofibroblast differentiation during pulmonary fibrosis [ 55 ]. Sun, Ni [ 56 ] found that by secreting S100A8/A9 and controlling FGF2-SOX9 signaling in fibroblasts as they age, G-MDSCs encourage cardiac fibrosis. Key molecular mechanisms allow MDSCs to suppress immunological responses include the elevation of STAT3 expression, development of ER stress, production of arginase 1, and expression of S100A8/A9 [ 57 ] ( Figure 3 ). An oncogene and latent transcription factor, Signal Transducer and Activator of Transcription 3 (STAT3) is triggered by a variety of cytokines and growth factors [ 58 ]. Papaioannou, Xu [ 59 ] mentioned that in human lung fibroblasts, collagen enhancer activation is not constrained by active STAT3. However, STAT3 activity must be over a particular threshold to enable activation of the COL1A2 enhancer and TGF-β signaling in fibroblasts. Chakraborty, Šumová [ 60 ] found that the interaction of JAK, SRC, c-ABL, and JNK kinases leads to STAT3 activation. The pro-fibrotic effects of TGF-β are less pronounced in fibroblasts lacking STAT3. In experimental animal models, skin fibrosis is reduced by pharmacological suppression of STAT3 or by STAT3-specific deletion. Heichler, Schmied [ 61 ] found that in colorectal carcinoma linked to colitis, inhibition of STAT3 activation in type I collagen-expressing fibroblasts may provide a feasible therapeutic target. Pedroza, To [ 62 ] mentioned that in mouse models of skin fibrosis, STAT3 inhibition with C188–9 reduced collagen deposition, myofibroblast accumulation, pro-fibrotic gene expression, and skin fibrosis. Additionally, they found that using C188–9 reduced in vitro dermal fibroblast production of fibrotic genes induced by IL-6 trans-signaling and TGF-β and that TGF-β caused STAT3 to become phosphotyrosylated in a way that was SMAD3 dependent. A STAT3 inhibitor called HJC0123 works by lowering STAT3 phosphorylation, nuclear translocation, and transcriptional activity. HJC0123 therapy has been shown to inhibit HSC activation and proliferation, induced its cell cycle arrest, promoted its apoptosis, and downregulated ECM proteins fibronectin and type I collagen production. Additionally, HJC0123 boosted the production of IL-6 and inhibited the phosphorylation of Smad2/3 caused by TGF-β [ 63 ]. STAT3 also regulates the activity of lysyl oxidase (LOX), a copper-dependent monoamine oxidase whose major role is to covalently cross-link collagen in the ECM, making it one of the key mediators in ECM deposition [ 64 ]. Park, Yoon [ 65 ] found that keloid fibroblast proliferation, migration, and collagen synthesis were severely inhibited by a JAK2/STAT3 inhibitor and a STAT3 siRNA. In vitro results from Matsui, Rhee [ 66 ] demonstrate that the response to IL-18 stimulation includes enhanced p-STAT3, SOCS3, α-SMA, collagen III expression, collagen synthesis, and TEC apoptosis; however, the reaction was considerably reduced in the presence of STAT3 inhibition. Arginase 1 (ARG1), one of the main enzymes that metabolizes arginine, is typically found in the cytoplasm while its isoform ARG2 is typically found in the mitochondria [ 67 ]. L-arginine is converted into L-ornithine and urea by the enzyme ARG1, which is produced by MDSCs and TAMs, and is essential for ammonia detoxification in the liver [ 68 ]. An ARG1-induced reduction of L-arginine reduces TCR chain expression, prevents T cell differentiation, arrests the T cell cycle, and reduces cytokine production. As a result, increased T cell-mediated anti-tumor responses are inhibited by higher ARG1 expression in the tumor microenvironment. Tumor cells may promote the growth of ARG1-expressing cells, such as tumor-associated macrophages (TAMs) and MDSCs, to elude immune surveillance [ 69 ]. Dias, Ciambarella [ 70 ] suggested that ARG1 helps with fibrogenesis in silicosis. Inactivating Arg1 in activated HSCs effectively reduced collagen synthesis but did not affect cell division. The downregulation of arginase-ornithine pathway expression following Tianlongkechuanling (TL) administration is a key mechanism and prospective treatment for pulmonary fibrosis using TL [ 71 ]. Grasemann, Dhaliwal [ 72 ] reported that treatment with amino-2-borono-6-hexanoic acid, an arginase inhibitor, stopped collagen deposition caused by bleomycin (BLM). In rat tracheal and lung fibroblasts cultured in a proline-free medium, Warnken, Haag [ 73 ] reported that inhibiting arginase reduced basal collagen I accumulation by about one-third and greatly attenuated the TGF-β1-induced increase in collagen accumulation. The Ca2 + -binding proteins S100A8 and S100A9, also known as MRP8 and MRP14, respectively, are members of the S100 family [ 74 ]. Due to their stability, they frequently exist as heterodimers and seldom as homodimers. In neutrophils and monocytes, S100A8/A9 is constitutively expressed as a Ca2 + sensor, taking a role in cytoskeleton reorganization and arachidonic acid metabolism. S100A8/A9 is actively produced during inflammation and plays a crucial role in regulating the inflammatory response by encouraging leukocyte recruitment and triggering cytokine secretion [ 75 ]. Araki, Kinoshita [ 76 ] reported that S100A8/A9 play a direct role in fibroblast and myofibroblast proliferation, collagen synthesis. Additionally, S100A8/A9 induced collagen synthesis by fibroblasts. In a mouse model of BLM-induced lung fibrosis, the anti-S100A8/A9 neutralizing antibody reduced the effects of S100A8/A9 on fibroblasts and slowed the progression of fibrosis. Through the production of the cytokine IL-17, Th17 cells, a specific subset of CD4 + T cells, are crucial in triggering inflammation in autoimmune diseases and infections. Various human malignancies have been discovered to contain Th17 cells [ 77 ]. Th17 cells differ from other immune system cells in that they can secrete IL-17A, IL-17F, IL-21, IL-22, and CCL20 [ 78 ]. TH17 were found to induce collagen synthesis and thus upregulation of UFs through secretion of IL-17, IL-22, and CCL20 ( Figure 4 ). A class of cytokines called IL-17 is involved in both acute and long-term inflammatory responses. It has been suggested that IL-17 has a key role in the initiation and development of UF since studies have demonstrated that it has a considerable impact on collagen accumulation from assembly to accumulation to ECM. Two pathways are activated when IL-17 binds to its receptor: (1) the canonical pathway, which involves activating the NF-kB, MAPK, and CCAAT-enhancer-binding protein (C/EBP) pathways, which activate downstream genes and proteins like pro-inflammatory genes and genes involved in tissue remodeling; and (2) the non-canonical pathway, which involves mRNA transcript stabilization. Interleukin-17 improves the stability of CXCL1 mRNA and consequently CXCL1 expression. As a result, neutrophils are recruited and triggered inflammation [ 79 ]. Through lysyl oxidase enzyme activation, IL-17 was reported to promote collagen assembly in cardiac fibrosis [ 80 ]. Additionally, it was discovered that IL-17 enhanced the stemness features of tumors and increased hMSC proliferation [ 81 ]. In a mouse fibrosis model, it was discovered that Th17 cells increased fibroblast proliferation, cytokine production, and induced skin and lung fibrosis [ 82 ]. Besides, IL-17A changed the fibroblast transcription model to tumorigenic phenotype [ 83 ]. A crucial regulator of epithelial homeostasis is the cytokine interleukin-22 (IL-22). IL-22 also contributed to regulation of epithelial cell development and permeability, formation of mucus and antimicrobial proteins (AMPs), and complement synthesis that has been linked to epithelial barrier [ 84 ]. In cultured normal human dermal fibroblasts, IL-22 dramatically boosted the expression of type I collagen protein without affecting its mRNA levels [ 85 ]. Liu, Shang [ 86 ] found that through the JAK/STAT3 signaling pathway, the IL-22/IL-22R1 signaling pathway induced MRC-5 cell proliferation and the production of COL11 and COL12 in MRC-5 cells. Wu, Tan [ 87 ] reported that in cardiac fibroblasts treated with TGF-β1, in vitro recombinant mouse IL-22 therapy increased the production of α-SMA, collagen I, and collagen III. Type I collagen and MMP-1, which are expressed in wounds involving keratinocytes, may be mediated by IL-22, which may promote scarless healing [ 88 ]. The sole chemokine known to interact with CC chemokine receptor 6 (CCR6) is CCL20. The ligand-receptor pair CCL20-CCR6 plays a role at skin and mucosal surfaces under homeostatic and inflammatory conditions, as well as in pathology, including cancer and rheumatoid arthritis. It is responsible for the chemoattraction of immature dendritic cells (DC), effector/memory T-cells, and B-cells [ 89 ]. Recombinant CCL20 promoted the expression of the collagen Type I alpha 2 gene from human skin fibroblasts. On the other hand, collagen type I gene and protein expression were significantly decreased when CCL20’s siRNA gene was knocked down [ 90 ]. Han, Zou [ 91 ] reported that lead acetate-induced fibroblast proliferation, hydroxyproline levels, the synthesis of TGF-β, and the expression of ECM-related proteins (Collagen I and fibronectin) which were all decreased by CCL20 knockdown. Beider, Abraham [ 92 ] showed that elevated dosages of CCL20 considerably increased cell adherence to collagen type I while just modestly increasing cell adhesion to fibronectin.