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.

Long-Term Outcomes of Cord Blood Transplantation from an HLA-Identical Sibling for Patients with Bone Marrow Failure Syndromes: A Report From Eurocord, Cord Blood Committee and Severe Aplastic Anemia Working Party of the European Society for Blood and Marrow Transplantation.

Cord blood transplantation (CBT) from HLA-identical siblings is an attractive option for patients with bone marrow failure (BMF) syndrome because of the low risk of graft-versus-host disease (GVHD) and the absence of risk to the donor. We analyzed outcomes of 117 patients with inherited or acquired BMF syndrome who received CBT from a related HLA-identical donor in European Society for Blood and Marrow Transplantation centers between 1988 and 2014. Ninety-seven patients had inherited and 20 patients acquired BMF syndrome. Eighty-two patients received a single cord blood (CB) unit, whereas 35 patients received a combination of CB and bone marrow cells from the same donor. Median age at CBT was 6.7 years, and median follow-up was 86.7 months. The cumulative incidence function (CIF) of neutrophil recovery was 88.8% (95% CI, 83.1% to 94.9%), 100-day CIF of grades II to IV acute GVHD was 15.2%, and 7-year CIF of chronic GVHD was 14.5%. Overall survival at 7 years was 87.9% (95% CI, 80.8% to 92.6%), 89% for inherited and 81% for acquired BMF syndromes (P = .66). Results of this study are consistent with outcomes of bone marrow transplantation shown by previous series in the same setting and indicate that in pediatric patients with BMF syndrome, CBT from an HLA-identical sibling donor is associated with excellent long-term outcomes and that collection of CB unit at birth of a new sibling is strongly recommended.