Both miR-17-5p and miR-20a alleviate suppressive potential of myeloid-derived suppressor cells by modulating STAT3 expression

Myeloid-derived suppressor cells (MDSCs) were one of the major components of the immune suppressive network. STAT3 has an important role in regulating the suppressive potential of MDSCs. In this study, we found that the expression of STAT3 could be modulated by both miR-17-5p and miR-20a. The transfection of miR-17-5p or miR-20a remarkably reduces the expression of reactive oxygen species and the production of H(2)O(2), which are regulated by STAT3. MDSCs transfected with miR-17-5p or miR-20a are less able to suppress Ag-specific CD4 and CD8 T cells. Importantly, both miR-17-5p and miR-20a alleviate the suppressive function of MDSCs in vivo. The expression of miR-17-5p and miR-20a in tumor-associated MDSCs was found to be lower than in Gr1(+)CD11b(+) cells isolated from the spleens of disease-free mice. Tumor-associated factor downregulates the expression of both miR-17-5p and miR-20a. The modulation of miR-17-5p and miR-20a expression may be important for the process by which patients with a tumor can overcome the immune tolerance mediated by MDSCs. Our results suggest that miR-17-5p and miR-20a could potentially be used for immunotherapy against diseases, especially cancer, by blocking STAT3 expression.     

Tumor-secreted miR-214 induces regulatory T cells: a major link between immune evasion and tumor growth

An increased population of CD4⁺CD25^highFoxp3⁺ regulatory T cells (Tregs) in the tumor-associated microenvironment plays an important role in cancer immune evasion. However, the underlying mechanism remains unclear. Here we observed an increased secretion of miR-214 in various types of human cancers and mouse tumor models. Tumor-secreted miR-214 was sufficiently delivered into recipient T cells by microvesicles (MVs). In targeted mouse peripheral CD4⁺ T cells, tumor-derived miR-214 efficiently downregulated phosphatase and tensin homolog (PTEN) and promoted Treg expansion. The miR-214-induced Tregs secreted higher levels of IL-10 and promoted tumor growth in nude mice. Furthermore, in vivo studies indicated that Treg expansion mediated by cancer cell-secreted miR-214 resulted in enhanced immune suppression and tumor implantation/growth in mice. The MV delivery of anti-miR-214 antisense oligonucleotides (ASOs) into mice implanted with tumors blocked Treg expansion and tumor growth. Our study reveals a novel mechanism through which cancer cell actively manipulates immune response via promoting Treg expansion.     

Mannan-Binding Lectin Deficiency Limits Inflammation-induced Myeloid-Derived Suppressor Cells Expansion via Modulating Tumor Necrosis Factor Alpha-triggered Apoptosis

Background: Mannan-binding lectin (MBL), a soluble pattern recognition molecule in the innate immune system, is reported to be associated with the function of immune cells. Myeloid-derived suppressor cells (MDSCs) are mainly characterized by immunosuppressive activities involving several inflammatory diseases such as cancer, infection, and arthritis. Some of the factors inducing their apoptosis are known, however, mechanisms have not been identified. The underlying impact of MBL on the MDSCs especially under inflammatory conditions remains unknown. This study was designed to investigate whether MBL affects MDSCs survival during inflammation conditions.Methods: WT and MBL-deficient (MBL^-/-) mice were induced on day 0 of the experiment by subcutaneous injection of complete Freund''s adjuvant and then injected with incomplete Freund''s adjuvant into the knee joint space under general anesthesia on day 14 to induce inflammatory arthritis. The proportions of MDSCs in the spleen and blood and the serum level of the inflammatory cytokines were measured. In vitro study, MDSCs were isolated from the bone marrow of WT and MBL^-/- mice and cultured in the presence of interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 5 days with or without tumor necrosis factor-alpha (TNF-α).Results: After adjuvant treatment, MBL^-/- mice had a significantly lower frequency of MDSCs as well as elevated serum inflammatory cytokines levels compared to WT mice. MBL deficiency markedly inhibited the MDSCs frequency from mice bone marrow induced by IL-6 and GM-CSF in the presence of TNF-α in vitro. Mechanistic studies established that MBL inhibited MDSCs apoptosis via down-regulation of TNF-α/tumor necrosis factor-alpha receptor 1 (TNFR1) signaling pathway and subsequent caspase 3-dependent manner.Conclusion: Mannan-binding lectin deficiency inhibits myeloid-derived suppressor cells expansion via modulating TNF-α triggered apoptosis.     

MicroRNA-494 is required for the accumulation and functions of tumor-expanded myeloid-derived suppressor cells via targeting of PTEN

Myeloid-derived suppressor cells (MDSCs) potently suppress the anti-tumor immune responses and also orchestrate the tumor microenvironment that favors tumor angiogenesis and metastasis. The molecular networks regulating the accumulation and functions of tumor-expanded MDSCs are largely unknown. In this study, we identified microRNA-494 (miR-494), whose expression was dramatically induced by tumor-derived factors, as an essential player in regulating the accumulation and activity of MDSCs by targeting of phosphatase and tensin homolog (PTEN) and activation of the Akt pathway. TGF-β1 was found to be the main tumor-derived factor responsible for the upregulation of miR-494 in MDSCs. Expression of miR-494 not only enhanced CXCR4-mediated MDSC chemotaxis but also altered the intrinsic apoptotic/survival signal by targeting of PTEN, thus contributing to the accumulation of MDSCs in tumor tissues. Consequently, downregulation of PTEN resulted in increased activity of the Akt pathway and the subsequent upregulation of MMPs for facilitation of tumor cell invasion and metastasis. Knockdown of miR-494 significantly reversed the activity of MDSCs and inhibited the tumor growth and metastasis of 4T1 murine breast cancer in vivo. Collectively, our findings reveal that TGF-β1-induced miR-494 expression in MDSCs plays a critical role in the molecular events governing the accumulation and functions of tumor-expanded MDSCs and might be identified as a potential target in cancer therapy.     

Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 suppresses tumor growth in breast cancer-bearing mice by negatively regulating myeloid-derived suppressor cell functions

Myeloid-derived suppressor cells (MDSCs) are one of the most important cell types that contribute to negative regulation of immune responses in the tumor microenvironment. Recently, aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1), a novel pleiotropic cytokine, was identified as an antitumor protein that inhibits angiogenesis and induces antitumor responses. However, the effect of AIMP1 on MDSCs in the tumor environment remains unclear. In the present study, we demonstrated that AIMP1 significantly inhibited tumor growth in 4T1 breast cancer-bearing mice and reduced MDSCs population of tumor sites and spleens of tumor-bearing mice. AIMP1 reduced expansion of MDSCs from bone marrow-derived cells in the tumor-conditioned media. AIMP1 also negatively regulated suppressive activities of MDSCs by inhibiting IL-6 and NO production, and Arg-1 expression. Furthermore, treatment of breast cancer-bearing mice with AIMP1 decreased the capacity of MDSCs to suppress T cell proliferation and Treg cell induction. Western blot and inhibition experiments showed that downregulation of MDSCs functions by AIMP1 may result from attenuated activation of STATs, Akt, and ERK. These findings indicate that AIMP1 plays an essential role in negative regulation of suppressive functions of MDSCs. Therefore, it has a significant potential as a therapeutic agent for cancer treatment.     

Functional changes in myeloid-derived suppressor cells (MDSCs) during tumor growth: FKBP51 contributes to the regulation of the immunosuppressive function of MDSCs

Myeloid-derived suppressor cells (MDSCs) are increased by tumor-derived factors and suppress anti-tumor immunity. MDSCs obtained at a late time point after tumor injection had stronger suppressive activity than MDSCs obtained at an early time point, as measured by T cell proliferation assays. To find factors in MDSCs that change during tumor growth, we analyzed gene expression profiles from MDSCs at different time points after tumor injection. We found that immune response-related genes were downregulated but protumor function-related genes were upregulated in both monocytic MDSCs (Mo-MDSCs) and polymorphonuclear granulocytic MDSCs (PMN-MDSCs) at the late time point. Among differentially expressed genes, FK506 binding protein 51 (FKBP51), which is a member of the immunophilin protein family and plays a role in immunoregulation, was increased in the Mo-MDSCs and PMN-MDSCs isolated from the late time points. Experiments using small interfering RNA and a chemical inhibitor of FKBP51 revealed that FKBP51 contributes to the regulation of the suppressive function of MDSCs by increasing inducible NO synthase, arginase-1, and reactive oxygen species levels and enhancing NF-κB activity. Collectively, our data suggest that FKBP51 is a novel molecule that can be targeted to regulate the immunosuppressive function of MDSCs.     

综述:髓系衍生的抑制性细胞与肿瘤免疫耐受关系的研究进展

髓系衍生的抑制性细胞(myeloid-derived suppressor cells,MDSCs),是在肿瘤等病理因素的作用下髓系细胞发生分化障碍所产生的不同阶段髓系祖细胞的集合,具有广谱而强大的免疫抑制功能,是免疫系统的重要负性调节组件之一.研究表明:肿瘤微环境中的多种细胞因子或生长因子可通过激活相应的信号通路促进MDSCs扩增及活化,MDSCs进而通过多种机制抑制包括T细胞在内的多种免疫细胞的功能而促进肿瘤个体免疫耐受的发生.临床研究表明:肿瘤患者体内MDSCs的水平与肿瘤临床病程进展密切相关,基于MDSCs的免疫治疗也有望成为肿瘤免疫治疗的新策略.本文主要介绍了肿瘤中MDSCs的表型鉴定、扩增及活化机制、发挥免疫抑制作用的途径及机制、肿瘤中MDSCs的临床意义以及本领域需要解决的问题,以期对MDSCs在肿瘤免疫耐受中的作用进展提供参考.     

Depletion of regulatory T cells facilitates growth of established tumors: a mechanism involving the regulation of myeloid-derived suppressor cells by lipoxin A4

Regulatory T cells (Tregs) are thought to facilitate tumor development by suppressing protective antitumor immune responses. However, recent clinical and laboratory studies show that Tregs are a favorable element against cancer. In this study, we provide evidence that Tregs have both promoting and inhibiting effects on tumors, depending on the stage of tumor development. By using 0.5 mg cyclophosphamide, we constructed a murine liver cancer model in which Tregs were continuously and selectively depleted. Under such conditions, we found that tumor growth was inhibited at early stages but accelerated later on. Analysis of the tumor microenvironment disclosed that long-term Treg depletion by 0.5 mg cyclophosphamide treatment induced Gr-1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs). Ablation of MDSCs by anti-Gr-1 Ab blocked Treg depletion-induced promotion of tumor growth. Furthermore, lipoxygenases 5 and 12, two enzymes participating in the biosynthesis of the lipid anti-inflammatory mediator lipoxin A(4), were upregulated or downregulated by Treg depletion or adoptive transfer. Correspondingly, the levels of lipoxin A(4) were increased or decreased. Lipoxin A(4) thus regulated the induction of MDSCs in response to Treg depletion. These findings suggest that Tregs may play different roles at different stages of tumor growth: promoting early and inhibiting late tumor growth. Our study also suggests that the interplay among Tregs, MDSCs, and lipoxin A(4) tunes the regulation of tumor-associated inflammation.     

Signaling pathways involved in MDSC regulation

The immune system has evolved mechanisms to protect the host from the deleterious effects of inflammation. The generation of immune suppressive cells like myeloid derived suppressor cells (MDSCs) that can counteract T cell responses represents one such strategy. There is an accumulation of immature myeloid cells or MDSCs in bone marrow (BM) and lymphoid organs under pathological conditions such as cancer. MDSCs represent a population of heterogeneous myeloid cells comprising of macrophages, granulocytes and dendritic cells that are at early stages of development. Although, the precise signaling pathways and molecular mechanisms that lead to MDSC generation and expansion in cancer remains to be elucidated. It is widely believed that perturbation of signaling pathways involved during normal hematopoietic and myeloid development under pathological conditions such as tumorogenesis contributes to the development of suppressive myeloid cells. In this review we discuss the role played by key signaling pathways such as PI3K, Ras, Jak/Stat and TGFb during myeloid development and how their deregulation under pathological conditions can lead to the generation of suppressive myeloid cells or MDSCs. Targeting these pathways should help in elucidating mechanisms that lead to the expansion of MDSCs in cancer and point to methods for eliminating these cells from the tumor microenvironment.     

miR-34a expands myeloid-derived suppressor cells via apoptosis inhibition

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population and show significant expansion under pathological conditions. microRNA plays important roles in many biological processes, whether microRNAs have a function in the expansion of MDSCs is still not very clear. In this study, miR-34a overexpression can induce the expansion of MDSCs in bone marrow chimera and transgenic mice model. The experimental results suggest that miR-34a inhibited the apoptosis of MDSCs but did not affect the proliferation of MDSCs. The distinct mRNA microarray profiles of MDSCs of wild type and miR-34a over-expressing MDSCs combined with the target prediction of miR-34a suggest that miR-34a may target genes such as p2rx7, Tia1, and plekhf1 to inhibit the apoptosis of MDSCs. Taken together, miR-34a contributes to the expansion of MDSCs by inhibiting the apoptosis of MDSCs.     

Serum inhibits the immunosuppressive function of myeloid-derived suppressor cells isolated from 4T1 tumor-bearing mice

As more groups investigate the role of myeloid-derived suppressor cells (MDSCs) in promoting the growth of primary tumors and distant tumor metastases, it is imperative to ensure the accurate detection and quantification of MDSC immunosuppression ex vivo. MDSCs are defined by their ability to suppress immune responses. Although different in vitro culture conditions have been used to study MDSCs, the effect of different culture conditions on MDSC immunosuppression is unknown. We therefore isolated MDSCs from the lungs and spleens of 4T1 murine mammary tumor-bearing mice and assayed MDSC-mediated suppression of T cell responses under different culture conditions. We found that 4T1-induced MDSCs effectively suppressed T cell proliferation under serum-free conditions, but not when fetal calf serum (FCS) was present. FCS neither altered the immunosuppressive activities of other myeloid cell types (i.e., peritoneal or tumor-associated macrophages) nor modified the susceptibility of T cells to myeloid cell-mediated suppression, but instead acted directly on 4T1-induced MDSCs to significantly reduce their immunosuppressive function. Importantly, we found that bovine serum albumin was a major contributor to the antagonistic effects of FCS on 4T1-induced MDSC immunosuppression by inhibiting reactive oxygen species production from MDSCs. This work reveals that in vitro culture conditions influence the immunosuppressive properties of MDSCs and highlights the importance of testing different culture conditions on MDSC phenotype to ensure that MDSC immunosuppression is not being masked. These data have important implications for the accurate detection and identification of MDSCs, as well as for determining the influence of MDSC-mediated immunosuppression on primary and metastatic tumor growth.     

Metabolic reprogramming of myeloid-derived suppressor cells in the context of organ transplantation

Myeloid-derived suppressor cells (MDSCs) are naturally occurring leukocytes that develop from immature myeloid cells under inflammatory conditions that were discovered initially in the context of tumor immunity. Because of their robust immune inhibitory activities, there has been growing interest in MDSC-based cellular therapies for transplant tolerance induction. Indeed, various pre-clinical studies have introduced in vivo expansion or adoptive transfer of MDSC as a promising therapeutic strategy leading to a profound extension of allograft survival due to suppression of alloreactive T cells. However, several limitations of cellular therapies using MDSCs remain to be addressed, including their heterogeneous nature and limited expansion capacity. Metabolic reprogramming plays a crucial role for differentiation, proliferation and effector function of immune cells. Notably, recent reports have focused on a distinct metabolic phenotype underlying the differentiation of MDSCs in an inflammatory microenvironment representing a regulatory target. A better understanding of the metabolic reprogramming of MDSCs may thus provide novel insights for MDSC-based treatment approaches in transplantation. In this review, we will summarize recent, interdisciplinary findings on MDSCs metabolic reprogramming, dissect the underlying molecular mechanisms and discuss the relevance for potential treatment approaches in solid-organ transplantation.     

Transgenic overexpression of the miR-200b/200a/429 cluster inhibits mammary tumor initiation

The miR-200 family consists of five members expressed as two clusters: miR-200c/141 cluster and miR-200b/200a/429 cluster. In the mammary gland, miR-200s maintain epithelial identity by decreasing the expression of mesenchymal markers leading to high expression of epithelial markers. While the loss of miR-200s is associated with breast cancer growth and metastasis the impact of miR-200 expression on mammary tumor initiation has not been investigated. Using mammary specific expression of the miR-200b/200a/429 cluster in transgenic mice, we found that elevated expression miR-200s could almost completely prevent mammary tumor development. Only 1 of 16 MTB-IGFIRba429 transgenic mice (expressing both the IGF-IR and miR-200b/200a/429 transgenes) developed a mammary tumor while 100% of MTB-IGFIR transgenic mice (expressing only the IGF-IR transgene) developed mammary tumors. RNA sequencing, qRT-PCR, and immunohistochemistry of mammary tissue from 55-day old mice found Spp1, Saa1, and Saa2 to be elevated in mammary tumors and inhibited by miR-200b/200a/429 overexpression. This study suggests that miR-200s could be used as a preventative strategy to protect women from developing breast cancer. One concern with this approach is the potential negative impact miR-200 overexpression may have on mammary function. However, transgenic overexpression of miR-200s, on their own, did not significantly impact mammary ductal development indicating the miR-200 overexpression should not significantly impact mammary function. Thus, this study provides the initial foundation for using miR-200s for breast cancer prevention and additional studies should be performed to identify strategies for increasing mammary miR-200 expression and determine whether miR-200s can prevent mammary tumor initiation by other genetic alterations.     

MiR-146a promotes the asymmetric division and inhibits the self-renewal ability of breast cancer stem-like cells via indirect upregulation of Let-7

MiR-146a could stimulate tumor growth or block tumor proliferation in systemic malignancies, referring to the specific downstream targeted gene. However, its roles in breast cancer stem-like cells (BrCSCs) are barely known. To dig out its mechanistic functions, we explored the indicative roles of miR-146 in preclinical study, regardless of the hormone receptor status, and the positive correlation between miR-146 and better prognosis was proved, as its correlation to Let-7c was. To uncover the implicated mechanisms, we first identified the suppressive role of miR-146a in stem cells’ renewal, which was achieved by promoting the asymmetric division of BrCSCs. Let-7c was previously revealed with its suppressive functions in stem-like cells expansion, and miR-146 was predicated and successfully proved to bind to and degrade the 3’UTR of LIN28, a maturation blocker of Let-7 family. Results further showed that miR-146a increased the Let-7c level through degrading LIN28, and LIN28 inhibition is required for miR-146a induction of asymmetric stem cells’ division. Moreover, Let-7 controlled Wnt signaling pathway activity could be strengthened due to the miR146 inhibition of H19, later of which was often activated in stem cells group with functional existence of Wnt signaling. H19 itself in turn formed the positive feedback regulation with Let-7. Our results suggested the miR-146a/LIN28/Wnt signaling circle in restraining the symmetric cells division, which was specifically referred to the controlling of the small circle of Let-7c and H19, and together, this dual axis could help to prohibit the stem cells expansion.     

Transgenic overexpression of the miR-200b/200a/429 cluster inhibits mammary tumor initiation

The miR-200 family consists of five members expressed as two clusters: miR-200c/141 cluster and miR-200b/200a/429 cluster. In the mammary gland, miR-200s maintain epithelial identity by decreasing the expression of mesenchymal markers leading to high expression of epithelial markers. While the loss of miR-200s is associated with breast cancer growth and metastasis the impact of miR-200 expression on mammary tumor initiation has not been investigated. Using mammary specific expression of the miR-200b/200a/429 cluster in transgenic mice, we found that elevated expression miR-200s could almost completely prevent mammary tumor development. Only 1 of 16 MTB-IGFIRba429 transgenic mice (expressing both the IGF-IR and miR-200b/200a/429 transgenes) developed a mammary tumor while 100% of MTB-IGFIR transgenic mice (expressing only the IGF-IR transgene) developed mammary tumors. RNA sequencing, qRT-PCR, and immunohistochemistry of mammary tissue from 55-day old mice found Spp1, Saa1, and Saa2 to be elevated in mammary tumors and inhibited by miR-200b/200a/429 overexpression. This study suggests that miR-200s could be used as a preventative strategy to protect women from developing breast cancer. One concern with this approach is the potential negative impact miR-200 overexpression may have on mammary function. However, transgenic overexpression of miR-200s, on their own, did not significantly impact mammary ductal development indicating the miR-200 overexpression should not significantly impact mammary function. Thus, this study provides the initial foundation for using miR-200s for breast cancer prevention and additional studies should be performed to identify strategies for increasing mammary miR-200 expression and determine whether miR-200s can prevent mammary tumor initiation by other genetic alterations.     

Myeloid-Derived Suppressor Cells in Murine Retrovirus-Induced AIDS Inhibit T- and B-Cell Responses In Vitro That Are Used To Define the Immunodeficiency

Myeloid-derived suppressor cells (MDSCs) have been characterized in several disease settings, especially in many tumor systems. Compared to their involvement in tumor microenvironments, however, MDSCs have been less well studied in their responses to infectious disease processes, in particular to retroviruses that induce immunodeficiency. Here, we demonstrate for the first time the development of a highly immunosuppressive MDSC population that is dependent on infection by the LP-BM5 retrovirus, which causes murine acquired immunodeficiency. These MDSCs express a cell surface marker signature (CD11b⁺ Gr-1⁺ Ly6C⁺) characteristic of monocyte-type MDSCs. Such MDSCs profoundly inhibit immune responsiveness by a cell dose- and substantially inducible nitric oxide synthase (iNOS)-dependent mechanism that is independent of arginase activity, PD-1–PD-L1 expression, and interleukin 10 (IL-10) production. These MDSCs display levels of immunosuppressive function in parallel with the extent of disease in LP-BM5-infected wild-type (w.t.) versus knockout mouse strains that are differentially susceptible to pathogenesis. These MDSCs suppressed not only T-cell but also B-cell responses, which are an understudied target for MDSC inhibition. The MDSC immunosuppression of B-cell responses was confirmed by the use of purified B responder cells, multiple B-cell stimuli, and independent assays measuring B-cell expansion. Retroviral load measurements indicated that the suppressive Ly6G^low/± Ly6C⁺ CD11b⁺-enriched MDSC subset was positive for LP-BM5, albeit at a significantly lower level than that of nonfractionated splenocytes from LP-BM5-infected mice. These results, including the strong direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression and, as this broadly suppressive function mirrors that of the LP-BM5-induced disease syndrome, support a possible pathogenic effector role for these retrovirus-induced MDSCs.     

Suppressive and Hypermethylated MicroRNAs in the Pathogenesis of Breast Cancer

MicroRNAs (miRNAs) are involved in oncogenesis by suppression of proto-oncogenes or tumor suppressive genes. This review presents data of suppressive miRNAs role in the mechanisms of occurrence and development of malignant tumors of breast cancer as the example—that is the most widespread oncopathology in women. Targets and functions of suppressive and antimetastatic miRNAs have been illustrated, as well as for suppressive miRNAs with an oncogenic potential (such as miR-200a, miR-200c) that appears probably owing to the ability of miRNA to interact with a variety of targets depending on the cellular content. Based on the published and the authors’ own data, the role of hypermethylation of promoter regions in inhibition of expression and regulatory function of miRNA genes in breast cancer was characterized. In conclusion, the authors pointed future prospects of clinical application of suppressive miRNAs in diagnostics and treatment of breast cancer.