Preclinical studies imply that surgery triggers inflammation that may entail tumor outgrowth and metastasis. The potential impact of surgery-induced inflammation in human pancreatic cancer is insufficiently explored. This study included 17 patients with periampullary cancer [pancreatic ductal adenocarcinoma (PDAC) n = 14, ampullary carcinoma n = 2, cholangiocarcinoma n = 1] undergoing major pancreatic cancer surgery with curative intent. We analyzed the potential impact of preoperative and postoperative immune phenotypes and function on postoperative survival with >30 months follow-up. The surgery entailed prompt expansion of monocytic myeloid-derived suppressor cells (M-MDSC) that generated NOX2-derived reactive oxygen species (ROS). Strong induction of immunosuppressive M-MDSC after surgery predicted poor postoperative survival and coincided with reduced functionality of circulating natural killer (NK) cells. The negative impact of surgery-induced M-MDSC on survival remained significant in separate analysis of patients with PDAC. M-MDSC-like cells isolated from patients after surgery significantly suppressed NK cell function ex vivo, which was reversed by inhibition of NOX2-derived ROS. High NOX2 subunit expression within resected tumors from patients with PDAC correlated with poor survival whereas high expression of markers of cytotoxic cells associated with longer survival. The surgery-induced myeloid inflammation was recapitulated in vivo in a murine model of NK cell-dependent metastasis. Surgical stress thus induced systemic accumulation of M-MDSC-like cells and promoted metastasis of NK cell-sensitive tumor cells. Genetic or pharmacologic suppression of NOX2 reduced surgery-induced inflammation and distant metastasis in this model. We propose that NOX2-derived ROS generated by surgery-induced M-MDSC may be targeted for improved outcome after pancreatic cancer surgery.
Pancreatic cancer surgery triggered pronounced accumulation of NOX2+ myeloid-derived suppressor cells that inhibited NK cell function and negatively prognosticated postoperative patient survival. We propose the targeting of M-MDSC as a conceivable strategy to reduce postoperative immunosuppression in pancreatic cancer.
© 2024 The Authors; Published by the American Association for Cancer Research.

Isolation of viable immune cells from tissues is critically important to characterize cellular and molecular processes during homeostasis and disease. Here, we provide an optimized protocol to achieve high yields of viable intestinal lamina propria mononuclear cells (LPMCs). We describe steps for intestinal tissue collection from humans and nonhuman primates, followed by mechanical disruption and enzymatic digestion. Furthermore, we detail characterization of the mononuclear phagocyte (MP) subtypes by flow cytometry analysis. The protocol is repeatable and scalable for downstream applications. For complete details on the use and execution of this protocol, please refer to Cavarelli et al. (2022).
Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.

The difficulty to unambiguously identify the various subsets of mononuclear phagocytes (MNPs) of the intestinal lamina propria has hindered our understanding of the initial events occurring after mucosal exposure to HIV-1. Here, we compared the composition and function of MNP subsets at steady-state and following ex vivo and in vivo viral exposure in human and macaque colorectal tissues. Combined evaluation of CD11c, CD64, CD103, and CX3CR1 expression allowed to differentiate lamina propria MNPs subsets common to both species. Among them, CD11c+ CX3CR1+ cells expressing CCR5 migrated inside the epithelium following ex vivo and in vivo exposure of colonic tissue to HIV-1 or SIV. In addition, the predominant population of CX3CR1high macrophages present at steady-state partially shifted to CX3CR1low macrophages as early as three days following in vivo SIV rectal challenge of macaques. Our analysis identifies CX3CR1+ MNPs as novel players in the early events of HIV-1 and SIV colorectal transmission.
© 2022 The Author(s).

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and different degrees of B cell compartment alteration. Memory B cell differentiation requires the orchestrated activation of several intracellular signaling pathways that lead to the activation of a number of factors, such as nuclear factor kappa B (NF-κB) which, in turn, promote transcriptional programs required for long-term survival. The aim of this study was to determine if disrupted B cell differentiation, survival and activation in B cells in CVID patients could be related to defects in intracellular signaling pathways. For this purpose, we selected intracellular readouts that reflected the strength of homeostatic signaling pathways in resting cells, as the protein expression levels of the Bcl-2 family which transcription is promoted by NF-κB. We found reduced Bcl-2 protein levels in memory B cells from CVID patients. We further explored the possible alteration of this crucial prosurvival signaling pathway in CVID patients by analysing the expression levels of mRNAs from anti-apoptotic proteins in naive B cells, mimicking T cell-dependent activation in vitro with CD40L and interleukin (IL)-21. BCL-XL mRNA levels were decreased, together with reduced levels of AICDA, after naive B-cell activation in CVID patients. The data suggested a molecular mechanism for this tendency towards apoptosis in B cells from CVID patients. Lower Bcl-2 protein levels in memory B cells could compromise their long-term survival, and a possible less activity of NF-κB in naive B cells, may condition an inabilityto increase BCL-XL mRNA levels, thus not promoting survival in the germinal centers.
© 2020 British Society for Immunology.

Multiple myeloma (MM) progression and myeloma-associated bone disease (MBD) are highly dependent on bone marrow mesenchymal stromal cells (MSCs). MM-MSCs exhibit abnormal transcriptomes, suggesting the involvement of epigenetic mechanisms governing their tumor-promoting functions and prolonged osteoblast suppression. Here, we identify widespread DNA methylation alterations of bone marrow-isolated MSCs from distinct MM stages, particularly in Homeobox genes involved in osteogenic differentiation that associate with their aberrant expression. Moreover, these DNA methylation changes are recapitulated in vitro by exposing MSCs from healthy individuals to MM cells. Pharmacological targeting of DNMTs and G9a with dual inhibitor CM-272 reverts the expression of hypermethylated osteogenic regulators and promotes osteoblast differentiation of myeloma MSCs. Most importantly, CM-272 treatment prevents tumor-associated bone loss and reduces tumor burden in a murine myeloma model. Our results demonstrate that epigenetic aberrancies mediate the impairment of bone formation in MM, and its targeting by CM-272 is able to reverse MBD.