Granulocytic MDSC with Deficient CCR5 Alleviates Lipogenesis and Inflammation in Nonalcoholic Fatty Liver Disease.

C-C chemokine receptor type 5 (CCR5) positively contributes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), a common metabolic liver disease associated with chronic inflammation. CCR5 signaling also facilitates the immunosuppressive activity of a group of immature myeloid cells known as granulocytic myeloid-derived suppressor cells (g-MDSCs). While both hepatocyte and g-MDSC express CCR5, how CCR5 coordinates these two distinct cell types in the hepatic microenvironment remains largely unknown. Here, we used in vivo and ex vivo approaches to define the molecular details of how CCR5 mediates the crosstalk between hepatocytes and g-MDSCs in a mouse model of NAFLD. Global CCR5-deficient mice exhibited more severe steatosis, increased hepatic gene expression of lipogenesis, and exacerbated liver damage in diet-induced obesity. Either NAFLD or CCR5-deficiency per se is causative for the increase of g-MDSCs. Purified g-MDSCs have a higher survival rate in the fatty liver microenvironment, and blockade of CCR5 significantly decreases g-MDSCs' expression of anti-inflammatory factors. On the other hand, the null of CCR5 signaling increases hepatocytes' expression of lipogenic genes in the NAFLD microenvironment. Most importantly, inhibiting g-MDSCs' CCR5 signaling in the fatty liver microenvironment dramatically reduces STAT3 signaling, lipogenic, and pro-inflammatory gene expression in primary hepatocytes. Adoptive cell transfer experiments further demonstrate that CCR5-deficient g-MDSCs mitigate hepatic lipogenic gene expression without facilitating pro-inflammatory cytokine production and liver damage in NAFLD mice. These results suggest that targeting g-MDSCs' CCR5 signaling might serve as a potential therapeutic strategy for NAFLD.

IFN-γ regulates survival and function of tumor-induced CD11b+ Gr-1high myeloid derived suppressor cells by modulating the anti-apoptotic molecule Bcl2a1.

Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b(+) Gr-1(high) granulocytic MDSCs. Coculture of CD11b(+) Gr-1(high) granulocytic MDSCs with antigen-stimulated T cells and simultaneous blockade of IFN-γ by the use of anti-IFN-γ blocking antibody, IFN-γ(-/-) effector T cells, IFN-γR(-/-) MDSCs or STAT1(-/-) MDSCs led to upregulation of Bcl2a1 in CD11b(+) Gr-1(high) cells, improved survival, and enhanced their suppressor function. Molecular studies revealed that GM-CSF released by antigen-stimulated CD8(+) T cells induced Bcl2a1 upregulation, which was repressed in the presence of IFN-γ by a direct interaction of phosphorylated STAT-1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN-γ/ STAT1-dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.

Elucidating granulocytic myeloid-derived suppressor cell heterogeneity during Staphylococcus aureus biofilm infection.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated immature myeloid cells with immunosuppressive activity that expand during chronic inflammation, such as cancer and prosthetic joint infection (PJI). Myeloid-derived suppressor cells can be broadly separated into 2 populations based on surface marker expression and function: monocytic myeloid-derived suppressor cells (M-MDSCs) and granulocytic myeloid-derived suppressor cells (G-MDSCs). Granulocytic myeloid-derived suppressor cells are the most abundant leukocyte infiltrate during PJI; however, how this population is maintained in vivo and cellular heterogeneity is currently unknown. In this study, we identified a previously unknown population of Ly6G+Ly6C+F4/80+MHCII+ MDSCs during PJI that displayed immunosuppressive properties ex vivo. We leveraged F4/80 and MHCII expression by these cells for further characterization using cellular indexing of transcriptomes and epitopes by sequencing, which revealed a distinct transcriptomic signature of this population. F4/80+MHCII+ MDSCs displayed gene signatures resembling G-MDSCs, neutrophils, and monocytes but had significantly increased expression of pathways involved in cytokine response/production, inflammatory cell death, and mononuclear cell differentiation. To determine whether F4/80+MHCII+ MDSCs represented an alternate phenotypic state of G-MDSCs, Ly6G+Ly6C+F4/80-MHCII- G-MDSCs from CD45.1 mice were adoptively transferred into CD45.2 recipients using a mouse model of PJI. A small percentage of transferred G-MDSCs acquired F4/80 and MHCII expression in vivo, suggesting some degree of plasticity in this population. Collectively, these results demonstrate a previously unappreciated phenotype of F4/80+MHCII+ MDSCs during PJI, revealing that a granulocytic-to-monocytic transition can occur during biofilm infection.

Longitudinal characterization of circulating neutrophils uncovers phenotypes associated with severity in hospitalized COVID-19 patients.

Mechanisms of neutrophil involvement in severe coronavirus disease 2019 (COVID-19) remain incompletely understood. Here, we collect longitudinal blood samples from 306 hospitalized COVID-19+ patients and 86 controls and perform bulk RNA sequencing of enriched neutrophils, plasma proteomics, and high-throughput antibody profiling to investigate relationships between neutrophil states and disease severity. We identify dynamic switches between six distinct neutrophil subtypes. At days 3 and 7 post-hospitalization, patients with severe disease display a granulocytic myeloid-derived suppressor cell-like gene expression signature, while patients with resolving disease show a neutrophil progenitor-like signature. Humoral responses are identified as potential drivers of neutrophil effector functions, with elevated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulin G1 (IgG1)-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirm that while patient-derived IgG antibodies induce phagocytosis in healthy donor neutrophils, IgA antibodies predominantly induce neutrophil cell death. Overall, our study demonstrates a dysregulated myelopoietic response in severe COVID-19 and a potential role for IgA-dominant responses contributing to mortality.

The Role of Myeloid-Derived Suppressor Cells in Tumor Growth and Metastasis.

Myeloid-derived suppressor cells (MDSCs) are immature bone marrow-derived suppressive cells that are an important component of the pathological immune response associated with cancer. Expansion of MDSCs has been linked to poor disease outcome and therapeutic resistance in patients with various malignancies, making these cells potential targets for next-generation treatment strategies. MDSCs are classified into monocytic (M-MDSC) and polymorphonuclear/granulocytic (PMN-MDSC) subtypes that undertake distinct and numerous roles in the tumor microenvironment or systemically to drive disease progression. In this chapter, we will discuss how MDSC subsets contribute to the growth of primary tumors and induce metastatic spread by suppressing the antitumor immune response, supporting cancer stem cell (CSC)/epithelial-to-mesenchymal transition (EMT) phenotypes and promoting angiogenesis. We will also summarize the signaling networks involved in the crosstalk between cancer cells and MDSCs that could represent putative immunotherapy targets.

Type I Interferon Promotes Antitumor T Cell Response in CRPC by Regulating MDSC.

BACKGROUND: Metastatic castration-resistant prostate cancer (CRPC) is the leading cause of death among prostate cancer patients. Here, our aim was to ascertain the immune regulatory mechanisms involved in CRPC development and identify potential immunotherapies against CRPC. METHODS: A CRPC model was established using Myc-CaP cells in immune-competent FVB mice following castration. The immune cell profile of the tumor microenvironment (TME) was analyzed during CRPC development. Different immunotherapies were screened in the CRPC tumor model, and their efficacies and underlying mechanisms were investigated in vitro and in vivo. RESULTS: During CRPC development, the proportion of granulocytic myeloid-derived suppressor cells (G-MDSCs) in the TME increased. Among the immunotherapies tested, IFNα was more effective than anti-PD-L1, anti-CTLA-4, anti-4-1BB, IL-2, and IL-9 in reducing Myc-CaP CRPC tumor growth. IFNα reduced the number of G-MDSCs both in vitro during differentiation and in vivo in CRPC mice. Furthermore, IFNα reduced the suppressive function of G-MDSCs on T cell proliferation and activation. CONCLUSION: G-MDSCs are crucial to effective immunotherapy against CRPC. Treatment with IFNα presents a promising therapeutic strategy against CRPC. Besides the direct inhibition of tumor growth and the promotion of T cell priming, IFNα reduces the number and the suppressive function of G-MDSCs and restores T cell activation.

Severe COVID-19 Is Characterized by an Impaired Type I Interferon Response and Elevated Levels of Arginase Producing Granulocytic Myeloid Derived Suppressor Cells.

COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19 that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1+ G-MDSC (Arg+G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg+G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.

Neutrophils mediate Th17 promotion in COVID-19 patients.

From the beginning of 2020, an urgent need to understand the pathophysiology of SARS-CoV-2 disease (COVID-19), much of which is due to dysbalanced immune responses, resonates across the world. COVID-19-associated neutrophilia, increased neutrophil-to-lymphocyte ratio, aberrant neutrophil activation, and infiltration of neutrophils into lungs suggest that neutrophils are important players in the disease immunopathology. The main objective of this study was to assess the phenotypic and functional characteristics of neutrophils in COVID-19 patients, with particular focus on the interaction between neutrophils and T cells. We hypothesize that the altered functional characteristics of COVID-19 patient-derived neutrophils result in skewed Th1/Th17 adaptive immune response, thus contributing to disease pathology. The expansion of G-MDSC and immature forms of neutrophils was shown in the COVID-19 patients. In the COVID-19 neutrophil/T cell cocultures, neutrophils caused a strong polarity shift toward Th17, and, conversely, a reduction of IFNγ-producing Th1 cells. The Th17 promotion was NOS dependent. Neutrophils, the known modulators of adaptive immunity, skew the polarization of T cells toward the Th17 promotion and Th1 suppression in COVID-19 patients, contributing to the discoordinated orchestration of immune response against SARS-CoV-2. As IL-17 and other Th17-related cytokines have previously been shown to correlate with the disease severity, we suggest that targeting neutrophils and/or Th17 represents a potentially beneficial therapeutic strategy for severe COVID-19 patients.

Intermittent fasting suppressed splenic CD205+ G-MDSC accumulation in a murine breast cancer model by attenuating cell trafficking and inducing apoptosis.

Immune-based interventions are the most promising approach for new cancer treatments to achieve long-term cancer-free survival. However, the expansion of myeloid-derived suppression cells (MDSCs) attenuates the therapeutic potential of immunotherapy. We recently showed that CD205+ granulocytic MDSCs (G-MDSCs), but not T cells, are sensitive to glucose deficiency. Intermittent fasting (IF) may inhibit the growth of malignant cells by reducing serum glucose levels, but little is known regarding the influence of IF on MDSC expansion. Herein, we observed that IF selectively inhibited splenic accumulation of CD205+ G-MDSCs in a 4T1 and 4T07 transplant murine breast cancer model. The efficiency of IF in suppressing tumor growth was comparable to that of docetaxel. Further examination revealed that CXCR4 expression was concentrated in CD205+ subsets of tumor-induced G-MDSCs. Downregulation of CXCR4 correlated with a reduction in CD205+ G-MDSC trafficking from bone marrow to the spleen under IF treatment. In addition, ex vivo culture assays showed that glucose deficiency and 2-deoxy-D-glucose (2DG) treatment selectively induced massive death of splenic CD205+ G-MDSCs. Interestingly, 2DG emulated the phenomena of IF selectively suppressing the accumulation of CD205+ G-MDSCs in the spleen, upregulating cleaved caspase 3 in the tumor, downregulating Ki67 in the lung, and retarding the growth of transplanted 4T1 and 4T07 murine breast tumors. These findings suggest that IF inhibited cell trafficking through the downregulation of CXCR4 and induced apoptosis by altering glucose metabolism; this, suppressed the accumulation of tumor-induced splenic CD205+ G-MDSCs and in turn enhanced antitumor immunity.

Chronic Inflammation Contributes to Tumor Growth: Possible Role of L-Selectin-Expressing Myeloid-Derived Suppressor Cells (MDSCs).

Recent data have demonstrated that chronic inflammation is a crucial component of tumor initiation and progression. We previously reported that immature myeloid-derived suppressor cells (MDSCs) with immunosuppressive activity toward effector T cells were expanded in experimental chronic inflammation. We hypothesized that elevated levels of MDSCs, induced by chronic inflammation, may contribute to the progression of tumor growth. Using the Ehrlich carcinoma animal model, we found increased tumor growth in mice with chronic adjuvant arthritis, which was accompanied by a persistent increase in the proportion of splenic monocytic and granulocytic MDSCs expressing CD62L (L-selectin), when compared to tumor mice without adjuvant arthritis. Depletion of inflammation-induced MDSCs resulted in decreased tumor growth. In vitro studies demonstrated that increased expression of CD62L by MDSCs was mediated by TNFα, elevated concentrations of which were found in tumor mice subjected to chronic inflammation. Moreover, the addition of exogenous TNFα markedly enhanced the suppressive activity of bone marrow-derived MDSCs, as revealed by the ability to impair the proliferation of CD8+ T cells in vitro. This study provides evidence that chronic inflammation may promote tumor growth via induction of CD62L expression by MDSCs that can facilitate their migration to tumor and lymph nodes and modulation of their suppressor activity.

Bacterial Killing Activity of Polymorphonuclear Myeloid-Derived Suppressor Cells Isolated From Tumor-Bearing Dogs.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are implicated in the progression and outcome of a variety of pathological states, from cancer to infection. Our previous work has identified three antimicrobial peptides differentially expressed by PMN-MDSCs compared to conventional neutrophils isolated from dogs, mice, and human patients with cancer. We therefore hypothesized that PMN-MDSCs in dogs with cancer possess antimicrobial activity. In the current work, we observed that exposure of PMN-MDSCs to Gram-negative bacteria (Escherichia coli) increased the expression of reactive oxygen species by the PMN-MDSCs, indicating that they are capable of initiating an anti-microbial response. Electron microscopy revealed that the PMN-MDSCs phagocytosed Gram-negative and Gram-positive (Staphylococcus aureus) bacterial species. Lysis of bacteria within some of the PMN-MDSCs suggested bactericidal activity, which was confirmed by the recovery of significantly lower numbers of bacteria of both species following exposure to PMN-MDSCs isolated from tumor-bearing dogs. Our data therefore indicate that PMN-MDSCs isolated from dogs with cancer, in common with PMNs, have phagocytic and bactericidal activity. This nexus of immunosuppressive and antimicrobial activity reveals a hitherto unrecognized function of MDSCs.

Neutrophil Heterogeneity in Cancer: From Biology to Therapies.

Neutrophils have been extensively described in the pathophysiology of autoimmune and infectious diseases. Accumulating evidence also suggests the important role of neutrophils in cancer progression through their interaction with cancer and immune cells in blood and in the tumor microenvironment (TME). Most studies have described neutrophils as key drivers of cancer progression, due to their involvement in various tumor promoting functions including proliferation, aggressiveness, and dissemination, as well as in immune suppression. However, such studies were focusing on late-stages of tumorigenesis, in which chronic inflammation had already developed. The role of tumor-associated neutrophils (TANs) at early stages of tumor development remains poorly described, though recent findings indicate that early-stage TANs may display anti-tumor properties. Beyond their role at tumor site, evidence supported by NLR retrospective studies and functional analyses suggest that blood neutrophils could also actively contribute to tumorigenesis. Hence, it appears that the phenotype and functions of neutrophils vary greatly during tumor progression, highlighting their heterogeneity. The origin of pro- or anti-tumor neutrophils is generally believed to arise following a change in cell state, from resting to activated. Moreover, the fate of neutrophils may also involve distinct differentiation programs yielding various subsets of pro or anti-tumor neutrophils. In this review, we will discuss the current knowledge on neutrophils heterogeneity across different tissues and their impact on tumorigenesis, as well as neutrophil-based therapeutic strategies that have shown promising results in pre-clinical studies, paving the way for the design of neutrophil-based next generation immunotherapy.

Granulocyte-like myeloid derived suppressor cells (G-MDSC) are increased in multiple myeloma and are driven by dysfunctional mesenchymal stem cells (MSC).

Granulocytic-Myeloid-derived suppressor cells (G-MDSC) are increased in Multiple Myeloma (MM) patients but the mechanisms of G-MDSC generation are still unknown. There are many evidences of the role of mesenchymal stem cells (MSC) in promoting MM cell growth, survival and drug-resistance. We here used a specific experimental model in vitro to evaluate the ability of MSC to induce G-MDSC. We found that although MSC derived from healthy donors (HD), MGUS and MM were able to generate the same amount of MDSC, only MM-MSC-educated G-MDSC exhibited suppressive ability. In addition, in comparison with MSC derived from HD, MM-MSC produce higher amount of immune-modulatory factors that could be involved in MDSC induction. Compared to G-MDSC obtained from co-culture models with MSC from healthy subjects, both MGUS and MM-MSC-educated G-MDSC showed increase of immune-modulatory factors. However, only MM-MSC educated G-MDSC 1) up-regulated immune-suppressive factors as ARG1 and TNFα, 2) expressed higher levels of PROK2, important in angiogenesis and inflammatory process, and 3) showed ability to digest bone matrix.Our data demonstrate that MM-MSC are functionally different from healthy subjects and MGUS-MSC, supporting an evolving concept regarding the contribution of MM-MSC to tumor development and progression.

Adjuvants and myeloid-derived suppressor cells: enemies or allies in therapeutic cancer vaccination.

Adjuvants are a critical but largely overlooked and poorly understood component included in vaccine formulations to stimulate and modulate the desired immune responses to an antigen. However, unlike in the protective infectious disease vaccines, adjuvants for cancer vaccines also need to overcome the effect of tumor-induced suppressive immune populations circulating in tumor-bearing individuals. Myeloid-derived suppressor cells (MDSC) are considered to be one of the key immunosuppressive populations that inhibit tumor-specific T cell responses in cancer patients. This review focuses on the different signals for the activation of the immune system induced by adjuvants, and the close relationship to the mechanisms of recruitment and activation of MDSC. This work explores the possibility that a cancer vaccine adjuvant may either strengthen or weaken the effect of tumor-induced MDSC, and the crucial need to address this in present and future cancer vaccines.

Anti-melanoma vaccines engineered to simultaneously modulate cytokine priming and silence PD-L1 characterized using ex vivo myeloid-derived suppressor cells as a readout of therapeutic efficacy.

Efficacious antitumor vaccines strongly stimulate cancer-specific effector T cells and counteract the activity of tumor-infiltrating immunosuppressive cells. We hypothesised that combining cytokine expression with silencing programmed cell death ligand 1 (PD-L1) could potentiate anticancer immune responses of lentivector vaccines. Thus, we engineered a collection of lentivectors that simultaneously co-expressed an antigen, a PD-L1-silencing shRNA, and various T cell-polarising cytokines, including interferon γ (IFNγ), transforming growth factor ß (TGFß) or interleukins (IL12, IL15, IL23, IL17A, IL6, IL10, IL4). In a syngeneic B16F0 melanoma model and using tyrosinase related protein 1 (TRP1) as a vaccine antigen, we found that simultaneous delivery of IL12 and a PD-L1-silencing shRNA was the only combination that exhibited therapeutically relevant anti-melanoma activities. Mechanistically, we found that delivery of the PD-L1 silencing construct boosted T cell numbers, inhibited in vivo tumor growth and strongly cooperated with IL12 cytokine priming and antitumor activities. Finally, we tested the capacities of our vaccines to counteract tumor-infiltrating myeloid-derived suppressor cell (MDSC) activities ex vivo. Interestingly, the lentivector co-expressing IL12 and the PD-L1 silencing shRNA was the only one that counteracted MDSC suppressive activities, potentially underlying the observed anti-melanoma therapeutic benefit. We conclude that (1) evaluation of vaccines in healthy mice has no significant predictive value for the selection of anticancer treatments; (2) B16 cells expressing xenoantigens as a tumor model are of limited value; and (3) vaccines which inhibit the suppressive effect of MDSC on T cells in our ex vivo assay show promising and relevant antitumor activities.