Myeloid-derived Suppressor Cells Suppressive Functions Research Articles

Targeting miR-223 enhances myeloid-derived suppressor cell suppressive activities in multiple sclerosis patients.

Multiple sclerosis (MS) is an incurable autoimmune inflammatory demyelinating disease of the central nervous system. Several MS medications can modify disease course through effects on adaptive immune cells, while drugs targeting innate immunity are under investigation. Myeloid-derived suppressor cells (MDSCs) which arise during chronic inflammation, are defined by their T-cell immunosuppressive functions. MiR-223 modulates myeloid cell maturation and expansion, including MDSCs. MDSCs isolated from healthy controls (HC) and people with MS (pwMS) were co-cultured with CD4+ T-cells to study their immunosuppressive activities in vitro. Cytokines and chemokines concentration were evaluated by Luminex assay in the serum of HC, pwMS, and other neuroinflammatory diseases and correlated with MDSC activities. MDSC suppressive functions are dysregulated in pwMS compared to HC, which was reversed by glucocorticoids (GC). GC specifically downregulated miR-223 levels in MDSCs and increased the expression of STAT3. In vitro assay showed that miR-223 inhibition enhanced MDSC suppressive activity, STAT3 dependently. By multiple linear regression analysis, we demonstrated that MDSC phosphorylated STAT3 was correlated with serum GM-CSF in HC and pwMS. These results suggest that miR-223 could be a therapeutic target for enhancing MDSC's suppressive activities as an alternative to GC.

Blockade of Myd88 signaling by a novel MyD88 inhibitor prevents colitis-associated colorectal cancer development by impairing myeloid-derived suppressor cells

SummaryBackground. In cancer, myeloid-derived suppressor cells (MDSCs) are known to escape the host immune system by developing a highly suppressive environment. However, little is known about the molecular mechanism behind MDSC-mediated tumor cell evasion of the immune system. Toll-like receptor (TLR) signaling elicited in the tumor microenvironment has the potential to induce MDSC differentiations in different organs. Therefore, MDSC elimination by blocking the action of myeloid differentiation factor 88 (MyD88), which is a key adaptor-signaling molecule that affects TLR activity, seems to be an ideal tumor immunotherapy. Previous studies have proven that blocking MyD88 signaling with a novel MyD88 inhibitor (TJ-M2010-5, synthesized by Zhou’s group) completely prevented colitis-associated colorectal cancer (CAC) development in mice. Methods. In the present study, we investigated the impact of the novel MyD88 inhibitor on the number, phenotype, and function of MDSC in the mice model of CAC. Results. We showed that CAC growth inhibition was involved in diminished MDSC generation, expansion, and suppressive function and that MDSC-mediated immune escape was dependent on MyD88 signaling pathway activation. MyD88 inhibitor treatment decreased the accumulation of CD11b+Gr1+ MDSCs in mice with CAC, thereby reducing cytokine (GM-CSF, G-CSF, IL-1β, IL-6 and TGF-β) secretion associated with MDSC accumulation, and reducing the expression of molecules (iNOS, Arg-1 and IDO) associated with the suppressive capacity of MDSCs. In addition, MyD88 inhibitor treatment reduced the differentiation of MDSCs from myeloid cells and the suppressive capacity of MDSCs on the proliferation of activated CD4+ T cells in vitro. Conclusion. MDSCs are primary cellular targets of a novel MyD88 inhibitor during CAC development. Our findings prove that MyD88 signaling is involved in the regulation of the immunosuppressive functions of MDSCs. The novel MyD88 inhibitor TJ-M2010-5 is a new and effective agent that modulates MyD88 signaling to overcome MDSC suppressive functions, enabling the development of successful antitumor immunotherapy.

Long Noncoding RNA RUNXOR Promotes Myeloid-Derived Suppressor Cell Expansion and Functions via Enhancing Immunosuppressive Molecule Expressions during Latent HIV Infection.

RUNX1 overlapping RNA (RUNXOR) is a long noncoding RNA and a key regulator of myeloid-derived suppressor cells (MDSCs) via targeting runt-related transcription factor 1 (RUNX1). We and others have previously reported MDSC expansion and inhibition of host immune responses during viral infections; however, the mechanisms regulating MDSC differentiation and suppressive functions, especially the role of RUNXOR-RUNX1 in the regulation of MDSCs in people living with HIV (PLHIV), remain unknown. In this study, we demonstrate that RUNXOR and RUNX1 expressions are upregulated in MDSCs that expand and accumulate in human PBMCs derived from PLHIV. We found that the upregulation of RUNXOR and RUNX1 is associated with the expressions of several key immunosuppressive molecules, including arginase 1, inducible NO synthase, STAT3, IL-6, and reactive oxygen species. RUNXOR and RUNX1 could positively regulate each other's expression and control the expressions of these suppressive mediators. Specifically, silencing RUNXOR or RUNX1 expression in MDSCs from PLHIV attenuated MDSC expansion and immunosuppressive mediator expressions, whereas overexpressing RUNXOR in CD33+ myeloid precursors from healthy subjects promoted their differentiation into MDSCs and enhanced the expression of these mediators. Moreover, loss of RUNXOR-RUNX1 function in MDSCs improved IFN-γ production from cocultured autologous CD4 T cells derived from PLHIV. These results suggest that the RUNXOR-RUNX1 axis promotes the differentiation and suppressive functions of MDSCs via regulating multiple immunosuppressive signaling molecules and may represent a potential target for immunotherapy in conjunction with antiviral therapy in PLHIV.

HCV-Associated Exosomes Upregulate RUNXOR and RUNX1 Expressions to Promote MDSC Expansion and Suppressive Functions through STAT3-miR124 Axis.

RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA and plays a pivotal role in the differentiation of myeloid cells via targeting runt-related transcription factor 1 (RUNX1). We and others have previously reported that myeloid-derived suppressor cells (MDSCs) expand and inhibit host immune responses during chronic viral infections; however, the mechanisms responsible for MDSC differentiation and suppressive functions, in particular the role of RUNXOR–RUNX1, remain unclear. Here, we demonstrated that RUNXOR and RUNX1 expressions are significantly upregulated and associated with elevated levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS) in MDSCs during chronic hepatitis C virus (HCV) infection. Mechanistically, we discovered that HCV-associated exosomes (HCV-Exo) can induce the expressions of RUNXOR and RUNX1, which in turn regulates miR-124 expression via STAT3 signaling, thereby promoting MDSC differentiation and suppressive functions. Importantly, overexpression of RUNXOR in healthy CD33+ myeloid cells promoted differentiation and suppressive functions of MDSCs. Conversely, silencing RUNXOR or RUNX1 expression in HCV-derived CD33+ myeloid cells significantly inhibited their differentiation and expressions of suppressive molecules and improved the function of co-cultured autologous CD4 T cells. Taken together, these results indicate that the RUNXOR–RUNX1–STAT3–miR124 axis enhances the differentiation and suppressive functions of MDSCs and could be a potential target for immunomodulation in conjunction with antiviral therapy during chronic HCV infection.

LncRNA HOTAIRM1 promotes MDSC expansion and suppressive functions through the HOXA1-miR124 axis during HCV infection

HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1) is a long non-coding RNA (lncRNA) that plays a pivotal role in regulating myeloid cell development via targeting HOXA1 gene expression. We and others have previously shown that myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature myeloid cells, expand during chronic viral (HCV, HIV) infections. However, the role of HOTAIRM1 in the development and suppression of MDSCs during viral infection remains unknown. In this study, we demonstrate that the expressions of HOTAIRM1 and its target HOXA1 are substantially upregulated to promote the expressions of immunosuppressive molecules, including arginase 1, inducible nitric oxide synthase, signal transducer and activator of transcription 3, and reactive oxygen species, in CD33+ myeloid cells derived from hepatitis C virus (HCV)-infected patients. We show that HCV-associated exosomes (HCV-Exo) can modulate HOTAIRM1, HOXA1, and miR124 expressions to regulate MDSC development. Importantly, overexpression of HOTAIRM1 or HOXA1 in healthy CD33+ myeloid cells promoted the MDSC differentiation and suppressive functions; conversely, silencing of HOTAIRM1 or HOXA1 expression in MDSCs from HCV patients significantly reduced the MDSC frequency and their suppressive functions. In essence, these results indicate that the HOTAIRM1-HOXA1-miR124 axis enhances the differentiation and suppressive functions of MDSCs and may be a potential target for immunomodulation in conjunction with antiviral therapy during chronic viral infection.

Long noncoding RNA HOTAIRM1 promotes myeloid-derived suppressor cell expansion and suppressive functions through up-regulating HOXA1 expression during latent HIV infection.

Myeloid-derived suppressor cells (MDSCs) contribute to HIV progression by impairing antiviral immunity; however, the mechanisms responsible for MDSC development during HIV infection are incompletely understood. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) is a long noncoding RNA (lncRNA) that plays a pivotal role in regulating myeloid cell development via targeting HOXA1. The role of HOTAIRM1--HOXA1 in the differentiation and functions of MDSCs during HIV infection remains unclear. In this study, we measured MDSC induction and suppressive functions by flow cytometry, RT-PCR, and co-culture experiments using CD33 myeloid cells derived from people living with HIV (PLHIV) on antiretroviral therapy (ART). We also manipulated the HOTAIRM1--HOXA1 axis in myeloid cells using knockdown and overexpression approaches. We demonstrate that HOTAIRM1 and HOXA1 expressions are reciprocally upregulated and are responsible for increased levels of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), signal transducer and activator of transcription 3 (STAT3), and reactive oxygen species (ROS), in CD33 myeloid cells derived from PLHIV on ART. We found that overexpression of HOTAIRM1 or HOXA1 in CD33 cells isolated from healthy individuals promoted the differentiation and suppressive functions of MDSCs, whereas silencing of HOTAIRM1 or HOXA1 expression in MDSCs derived from PLHIV significantly inhibited the frequency of MDSCs and expressions of the immunosuppressive molecules and reduced their immunosuppressive effects on T cells. These results indicate that the HOTAIRM1--HOXA1 axis enhances differentiation and suppressive functions of MDSCs and could be a potential therapeutic target for immunomodulation during latent HIV infection.

LncRNA HOTAIRM1 promotes the immunosuppressive functions of MDSCs through upregulating HOXA1 expression during chronic HCV infection

Abstract HOXA transcript antisense RNA myeloid-specific 1 (HOTAIRM1) is a long non-coding RNA (lncRNA) that is critical for the myeloid cell development by targeting HOXA1. We have previously shown that myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that have immunosuppressive functions. However, the role and mechanisms of HOTAIRM1 in the differentiation and function of MDSCs in the setting of viral infection remain elusive. In this study, we demonstrated that HOTAIRM1 and its target gene HOXA1 were upregulated and closely associated with the expression of immunosuppressive molecules, such as arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), and signal transducer and activator of transcription 3 (STAT3) in myeloid cells derived from individuals with chronic hepatitis C virus (HCV) infection. We further revealed that HCV-associated exosomes could induce HATAIRM1 and HOXA1 expression via the miRNA-mediated signaling pathway. Importantly, overexpression of HOTAIRM1 or HOXA1 in healthy myeloid cells promoted MDSC differentiation and suppressive functions, evidenced by an increased frequency of MDSCs, expression of immunosuppressive molecules, and inhibition of co-cultured CD4 T cells to produce IFN-γ. Conversely, silencing HOTAIRM1 or HOXA1 expression in HCV myeloid cells partially restored the frequencies of MDSCs, the expressions of their suppressive molecule, and the IFN-γ production in co-cultured T cells. Taken together, this study reveals that HOTAIRM1-HOXA1 enhances the immunosuppressive function of MDSCs and may be a potential target for immunomodulation in conjunction with antiviral therapy during chronic viral infection.

The Adaptor Protein CARD9 Protects against Colon Cancer by Restricting Mycobiota-Mediated Expansion of Myeloid-Derived Suppressor Cells

The adaptor protein CARD9 links detection of fungi by surface receptors to the activation of the NF-κB pathway. Mice deficient in CARD9 exhibit dysbiosis and are more susceptible to colitis. Here we examined the impact of Card9 deficiency in the development of colitis-associated colon cancer (CAC). Treatment of Card9-/- mice with AOM-DSS resulted in increased tumor loads as compared to WT mice and in the accumulation of myeloid-derived suppressor cells (MDSCs) in tumor tissue. The impaired fungicidal functions of Card9-/- macrophages led to increased fungal loads and variation in the overall composition of the intestinal mycobiota, with a notable increase in C.tropicalis. Bone marrow cells incubated with C.tropicalis exhibited MDSC features and suppressive functions. Fluconazole treatment suppressed CAC in Card9-/- mice and was associated with decreased MDSC accumulation. The frequency of MDSCs in tumor tissues of colon cancer patients correlated positively with fungal burden, pointing to the relevance of this regulatory axis in human disease.

Early Expansion of Circulating Granulocytic Myeloid-derived Suppressor Cells Predicts Development of Nosocomial Infections in Patients with Sepsis.

Sepsis induces a sustained immune dysfunction responsible for poor outcome and nosocomial infections. Myeloid-derived suppressor cells (MDSCs) described in cancer and inflammatory processes may be involved in sepsis-induced immune suppression, but their clinical impact remains poorly defined. To clarify phenotype, suppressive activity, origin, and clinical impact of MDSCs in patients with sepsis. Peripheral blood transcriptomic analysis was performed on 29 patients with sepsis and 15 healthy donors. A second cohort of 94 consecutive patients with sepsis, 11 severity-matched intensive care patients, and 67 healthy donors was prospectively enrolled for flow cytometry and functional experiments. Genes involved in MDSC suppressive functions, including S100A12, S100A9, MMP8, and ARG1, were up-regulated in the peripheral blood of patients with sepsis. CD14posHLA-DRlow/neg monocytic (M)-MDSCs were expanded in intensive care unit patients with and without sepsis and CD14negCD15pos low-density granulocytes/granulocytic (G)-MDSCs were more specifically expanded in patients with sepsis (P < 0.001). Plasma levels of MDSC mediators S100A8/A9, S100A12, and arginase 1 were significantly increased. In vitro, CD14pos- and CD15pos-cell depletion increased T-cell proliferation in patients with sepsis. G-MDSCs, made of immature and mature granulocytes expressing high levels of degranulation markers, were specifically responsible for arginase 1 activity. High initial levels of G-MDSCs, arginase 1, and S100A12 but not M-MDSCs were associated with subsequent occurrence of nosocomial infections. M-MDSCs and G-MDSCs strongly contribute to T-cell dysfunction in patients with sepsis. More specifically, G-MDSCs producing arginase 1 are associated with a higher incidence of nosocomial infections and seem to be major actors of sepsis-induced immune suppression.

Abstract 4063: The oncogenic cell cycle-related kinase promotes tumor immune escape via modulation of myeloid-derived suppressor cells in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a sexually dimorphic cancer significantly associated with elevated levels of male sex hormone androgen and pro-inflammatory cytokine interleukin-6 (IL-6) (ref 1). Our previous genome-wide location and functional analysis has pinpointed cell cycle-related kinase (CCRK) as a critical mediator of androgen receptor (AR) oncogenic activity in HCC through a self-reinforcing circuitry involving β-catenin and the Polycomb repressor enhancer of zeste 2 (EZH2) (ref 2-4). As these key circuit players have also been shown to trigger inflammatory response and aggressiveness of HCC cells (ref 5,6), we aimed to investigate whether CCRK modulates tumor microenvironment during hepatocarcinogenesis. Expression profiling of 16 pro-inflammatory cytokines and chemokines revealed that ectopic expression of wild-type but not kinase-defective CCRK significantly induced the production of IL-6, IL-1β, tumor necrosis factor-α and cyclooxygenase 2 in immortal hepatic and HCC cells. Rescue experiments showed that CCRK-induced IL-6 production was dependent on EZH2 up-regulation and subsequent nuclear factor-κB activation. Based on the causal link between IL-6 and myeloid-derived suppressor cells (MDSCs), we next examined the relationship between CCRK and MDSCs in vitro and in vivo. By co-culturing CCRK-expressed or silenced hepatic and HCC cell lines with peripheral blood mononuclear cells (PBMCs) isolated from healthy donors, we found that CCRK stimulated the expansion of Lin-CD33+CD11b+HLA-DR-MDSCs from PBMCs by flow cytometry analysis. The induced MDSCs also showed higher suppressive activity against CD3+ T cell proliferation. In a high-fat diet-fed mouse model exposed to low-dose carcinogen diethylnitrosamine, administration of lentivirus expressing short-hairpin (sh)RNA against Ccrk significantly reduced &amp;gt;70% obesity-promoted tumor multiplicity and size compared to mice treated with control shRNA (p&amp;lt;0.01). Notably, Ccrk down-regulation also significantly reduced the population of circulating CD11b+Gr-1intMDSCs in the mice (p&amp;lt;0.05). In summary, our findings demonstrate that the oncogenic CCRK in hepatic cells can promote MDSC expansion and suppressive functions possibly via IL-6 production. Such tumor-stromal cell interaction provides survival advantage of cancer cells via immune escape. In conclusion, CCRK not only functions as a signaling hub but also an immunoregulator, thus representing a new cancer immunotherapy target.

Abstract A29: MIF controls human myeloid-derived immunosuppressive phenotype/function in melanoma educated CD14+ monocytes

Abstract Tumors suppress the host immune system by employing a variety of cellular immune modulators such as regulatory T cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). Recently, MDSCs have been recognized as critically important cellular immune modulators that serve to suppress both innate and adaptive immune responses in a wide variety of human malignancies. Macrophage migration inhibitory factor (MIF) is one the oldest cytokine activities described and is an important determinant of both innate and adaptive immune responses. Recent studies by our group revealed that MDSCs isolated from melanoma-bearing MIF-deficient mice are substantially less immunosuppressive than those isolated from MIF wildtype mice and these phenotypes correspond to significantly reduced primary and metastatic melanoma growth and progression. Importantly, 4-iodo-6-phenylpyrimidine (4-IPP – our previously discovered small MIF inhibitor) fully recapitulates MIF-deficiency in vitro and in vivo and serves to attenuate MDSC immunosuppression and melanoma disease progression in mice. To investigate whether MIF participates in human melanoma-induced MDSC differentiation and/or suppressive functions, an in vitro MDSC induction model was established using normal, primary human monocytes co-cultured with human melanoma cell lines in the presence or absence of the MIF antagonist, 4-IPP. Our studies suggest that the induction of CD14+/CD33+/HLADRlo/- myeloid-suppressive cells recently shown to represent the bulk of immunosuppressive activity in late stage melanoma patients is largely dependent on MIF. Importantly, small molecule inhibition of MIF in these MDSC-like cells almost completely abolishes their ability to suppress proliferation and IFN-gamma production by autologous T cells. Further, MIF-dependent suppressive activity of MDSCs is regulated via an NADPH oxidase induced ROS-mediated signaling mechanism. Finally, we demonstrate that small molecule inhibitors of MIF attenuate the suppressive properties of circulating CD14+/HLADRlo/- MDSCs isolated from stage III/IV melanoma patients confirming MIF as a critical mediator of MDSC-dependent immunosuppression in patients with advanced stage melanoma. These studies using human, clinically relevant experimental melanoma models lend strong support to the rationale that therapeutic inhibition of MIF in human melanoma MDSCs is a novel and clinically viable approach to enhancing anti-tumor T cell immunity in advanced melanoma patients. Citation Format: Kavitha Yaddanapudi, Beatriz E. Rendon, Gwyneth J. Lamont, John W. Eaton, Robert A. Mitchell. MIF controls human myeloid-derived immunosuppressive phenotype/function in melanoma educated CD14+ monocytes. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A29. doi:10.1158/1538-7445.CHTME14-A29

Role of myeloid-derived suppressor cells in autoimmune disease

Myeloid-derived suppressor cells (MDSCs) represent an important class of immunoregulatory cells that can be activated to suppress T cell functions. These MDSCs can inhibit T cell functions through cell surface interactions and the release of soluble mediators. MDSCs accumulate in the inflamed tissues and lymphoid organs of patients with autoimmune diseases. Much of our knowledge of MDSC function has come from studies involving cancer models, however many recent studies have helped to characterize MDSC involvement in autoimmune diseases. MDSCs are a heterogeneous group of immature myeloid cells with a number of different functions for the suppression of T cell responses. However, we have yet to fully understand their contributions to the development and regulation of autoimmune diseases. A number of studies have described beneficial functions of MDSCs during autoimmune diseases, and thus there appears to be a potential role for MDSCs in the treatment of these diseases. Nevertheless, many questions remain as to the activation, differentiation, and inhibitory functions of MDSCs. This review aims to summarize our current knowledge of MDSC subsets and suppressive functions in tissue-specific autoimmune disorders. We also describe the potential of MDSC-based cell therapy for the treatment of autoimmune diseases and note some of hurdles facing the implementation of this therapy.

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.