The Cross-talk Between Intestinal Microbiota and MDSCs Fuels Colitis-associated Cancer Development.

Intestinal chronic inflammation is associated with microbial dysbiosis and accumulation of various immune cells including myeloid-derived suppressor cells (MDSC), which profoundly impact the immune microenvironment, perturb homeostasis and increase the risk to develop colitis-associated colorectal cancer (CAC). However, the specific MDSCs-dysbiotic microbiota interactions and their collective impact on CAC development remain poorly understood. In this study, using a murine model of CAC, we demonstrate that CAC-bearing mice exhibit significantly elevated levels of highly immunosuppressive MDSCs, accompanied by microbiota alterations. Both MDSCs and bacteria that infiltrate the colon tissue and developing tumors can be found in close proximity, suggesting intricate MDSC-microbiota cross-talk within the tumor microenvironment. To investigate this phenomenon, we employed antibiotic treatment to disrupt MDSC-microbiota interactions. This intervention yielded a remarkable reduction in intestinal inflammation, decreased MDSC levels, and alleviated immunosuppression, all of which were associated with a significant reduction in tumor burden. Furthermore, we underscore the causative role of dysbiotic microbiota in the predisposition toward tumor development, highlighting their potential as biomarkers for predicting tumor load. We shed light on the intimate MDSCs-microbiota cross-talk, revealing how bacteria enhance MDSC suppressive features and activities, inhibit their differentiation into mature beneficial myeloid cells, and redirect some toward M2 macrophage phenotype. Collectively, this study uncovers the role of MDSC-bacteria cross-talk in impairing immune responses and promoting tumor growth, providing new insights into potential therapeutic strategies for CAC. SIGNIFICANCE: MDSCs-dysbiotic bacteria interactions in the intestine play a crucial role in intensifying immunosuppression within the CAC microenvironment, ultimately facilitating tumor growth, highlighting potential therapeutic targets for improving the treatment outcomes of CAC.

Functional Assays Evaluating Immunosuppression Mediated by Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation, characterizing an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases, and induce immunosuppression. The suppressive effects of MDSCs on the immune system are studied mainly when focusing on their features, functions, and impact on target cells such as T cells, natural killer cells, and B cells, among others. Herein, we describe methods for the analysis of MDSC immunosuppressive features and functions, measuring different mediators that contribute to their activities and how they impact on T cell function. The protocols described are a continuation to those in a companion Current Protocols article by Reuven et al. (2022), which uses a generated single-cell suspension and isolated cells to test their activity. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Evaluating MDSC suppressive features Alternate Protocol 1: Dichlorofluorescein diacetate-based reactive oxygen species detection Support Protocol 1: Detection of nitric oxide secretion Support Protocol 2: Measurement of arginase activity Basic Protocol 2: Evaluating MDSC suppressive function Alternate Protocol 2: In vitro effects of MDSCs on expression of T cell receptor complex during activation Support Protocol 3: Effect of MDSCs on interferon γ production Basic Protocol 3: Effect of MDSCs on T cell proliferation Basic Protocol 4: Effect of MDSCs on T cell cytotoxic activity Alternate Protocol 3: In vivo cytotoxicity assay Basic Protocol 5: Analysis of MDSC differentiation.

Phenotypic Characterization and Isolation of Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are heterogenous populations of immature myeloid cells that can be divided into two main subpopulations, polymorphonuclear (PMN) MDSCs and monocytic (M) MDSCs. These cells accumulate during chronic inflammation and induce immunosuppression evident in an array of pathologies such as cancer, inflammatory bowel disease, and infectious and autoimmune diseases. Herein, we describe methods to isolate and characterize MDSCs from various murine tissue, as well as to phenotype blood-derived MDSCs from patients. The protocols describe methods for isolation of total MDSCs and their subpopulations, for characterization, and for evaluation of their distribution within tissue, as well as for assessing their maturation stage by flow cytometry, immunofluorescence analyses, and Giemsa staining. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single-cell suspension generation from different tissue Alternate Protocol 1: Single-cell suspension generation from subcutaneous melanoma tumors Basic Protocol 2: Characterization of MDSC phenotype Basic Protocol 3: Cell separation using magnetic beads: Separating pan-MDSCs or PMN-MDSC and M-MDSC subpopulations Alternate Protocol 2: Staining and preparing MDSCs for sorting Support Protocol: PMN-MDSC and M-MDSC gating strategy in mouse Basic Protocol 4: Immunofluorescence analysis of MDSCs Basic Protocol 5: Handling human blood samples and characterizing human MDSCs Alternate Protocol 3: Flow cytometry staining of thawed human whole blood samples.

Specific inflammatory osteoclast precursors induced during chronic inflammation give rise to highly active osteoclasts associated with inflammatory bone loss.

Elevated osteoclast (OC) activity is a major contributor to inflammatory bone loss (IBL) during chronic inflammatory diseases. However, the specific OC precursors (OCPs) responding to inflammatory cues and the underlying mechanisms leading to IBL are poorly understood. We identified two distinct OCP subsets: Ly6ChiCD11bhi inflammatory OCPs (iOCPs) induced during chronic inflammation, and homeostatic Ly6ChiCD11blo OCPs (hOCPs) which remained unchanged. Functional and proteomic characterization revealed that while iOCPs were rare and displayed low osteoclastogenic potential under normal conditions, they expanded during chronic inflammation and generated OCs with enhanced activity. In contrast, hOCPs were abundant and manifested high osteoclastogenic potential under normal conditions but generated OCs with low activity and were unresponsive to the inflammatory environment. Osteoclasts derived from iOCPs expressed higher levels of resorptive and metabolic proteins than those generated from hOCPs, highlighting that different osteoclast populations are formed by distinct precursors. We further identified the TNF-α and S100A8/A9 proteins as key regulators that control the iOCP response during chronic inflammation. Furthermore, we demonstrated that the response of iOCPs but not that of hOCPs was abrogated in tnf-α-/- mice, in correlation with attenuated IBL. Our findings suggest a central role for iOCPs in IBL induction. iOCPs can serve as potential biomarkers for IBL detection and possibly as new therapeutic targets to combat IBL in a wide range of inflammatory conditions.

The diverse roles of myeloid derived suppressor cells in mucosal immunity.

The mucosal immune system plays a vital role in protecting the host from the external environment. Its major challenge is to balance immune responses against harmful and harmless agents and serve as a 'homeostatic gate keeper'. Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of undifferentiated cells that are characterized by an immunoregulatory and immunosuppressive phenotype. Herein we postulate that MDSCs may be involved in shaping immune responses related to mucosal immunity, due to their immunomodulatory and tissue remodeling functions. Until recently, MDSCs were investigated mainly in cancerous diseases, where they induce and contribute to an immunosuppressive and inflammatory environment that favors tumor development. However, it is now becoming clear that MDSCs participate in non-cancerous conditions such as chronic infections, autoimmune diseases, pregnancy, aging processes and immune tolerance to commensal microbiota at mucosal sites. Since MDSCs are found in the periphery only in small numbers under normal conditions, their role is highlighted during pathologies characterized by acute or chronic inflammation, when they accumulate and become activated. In this review, we describe several aspects of the current knowledge characterizing MDSCs and their involvement in the regulation of the mucosal epithelial barrier, their crosstalk with commensal microbiota and pathogenic microorganisms, and their complex interactions with a variety of surrounding regulatory and effector immune cells. Finally, we discuss the beneficial and harmful outcomes of the MDSC regulatory functions in diseases affecting mucosal tissues. We wish to illuminate the pivotal role of MDSCs in mucosal immunity, the limitations in our understanding of all the players and the intricate challenges stemming from the complex interactions of MDSCs with their environment.