From Monocytes to M1/M2 Macrophages: Phenotypical vs. Functional Differentiation

Studies on monocyte and macrophage biology and differentiation have revealed the pleiotropic activities of these cells. Macrophages are tissue sentinels that maintain tissue integrity by eliminating/repairing damaged cells and matrices. In this M2-like mode they can also promote tumor growth. Conversely, M1-like macrophages are key effector cells for the elimination of pathogens, virally infected, and cancer cells. Macrophage differentiation from monocytes occurs in the tissue in concomitance with the acquisition of a functional phenotype that depends on microenvironmental signals, thereby accounting for the many and apparently opposed macrophage functions. Many questions arise. When monocytes differentiate into macrophages in a tissue (concomitantly adopting a specific functional program, M1 or M2), do they all die during the inflammatory reaction, or do some of them survive? Do those that survive become quiescent tissue macrophages, able to react as naïve cells to a new challenge? Or, do monocyte-derived tissue macrophages conserve a memory of their past inflammatory activation? This review will address some of these important questions under the general framework of the role of monocytes and macrophages in the initiation, development, resolution and chronicization of inflammation

The SARS-CoV-2 Nucleoprotein Induces Innate Memory in Human Monocytes

The interaction of SARS-CoV-2 with the human immune system is at the basis of the positive or negative outcome of the infection. Monocytes and macrophages, which are major innate immune/inflammatory effector cells, are not directly infected by SARS-CoV-2, however they can react to the virus and mount a strong reaction. Whether this first interaction and reaction may bias innate reactivity to re-challenge, a phenomenon known as innate memory, is currently unexplored and may be part of the long-term sequelae of COVID-19. Here, we have tested the capacity of SARS-CoV-2 and some of its proteins to induce innate memory in human monocytes in vitro. Our preliminary results show that the Spike protein subunits S1 and S2 and the entire heat-inactivated virus have no substantial effect. Conversely, monocytes pre-exposed to the nucleocapsid N protein react to subsequent viral or bacterial challenges with an increased production of anti-inflammatory IL-1Ra, a response profile suggesting a milder response to new infections

Mucosal delivery of anti-inflammatory IL-1Ra by sporulating recombinant bacteria

BACKGROUND: Mucosal delivery of therapeutic protein drugs or vaccines is actively investigated, in order to improve bioavailability and avoid side effects associated with systemic administration. Orally administered bacteria, engineered to produce anti-inflammatory cytokines (IL-10, IL-1Ra), have shown localised ameliorating effects in inflammatory gastro-intestinal conditions. However, the possible systemic effects of mucosally delivered recombinant bacteria have not been investigated. RESULTS: B. subtilis was engineered to produce the mature human IL-1 receptor antagonist (IL-1Ra). When recombinant B. subtilis was instilled in the distal colon of rats or rabbits, human IL-1Ra was found both in the intestinal lavage and in the serum of treated animals. The IL-1Ra protein in serum was intact and biologically active. IL-1-induced fever, neutrophilia, hypoglycemia and hypoferremia were inhibited in a dose-dependent fashion by intra-colon administration of IL-1Ra-producing B. subtilis. In the mouse, intra-peritoneal treatment with recombinant B. subtilis could inhibit endotoxin-induced shock and death. Instillation in the rabbit colon of another recombinant B. subtilis strain, which releases bioactive human recombinant IL-1β upon autolysis, could induce fever and eventually death, similarly to parenteral administration of high doses of IL-1β. CONCLUSIONS: A novel system of controlled release of pharmacologically active proteins is described, which exploits bacterial autolysis in a non-permissive environment. Mucosal administration of recombinant B. subtilis causes the release of cytoplasmic recombinant proteins, which can then be found in serum and exert their biological activity in vivo systemically

Impact of engineered nanoparticles in initiating or modulating pathology-related Inflammation

The possibility that nanomaterials could perturb the normal course of an inflammatory response is a key issue when assessing nano-immunosafety. The alteration of the normal progress of an inflammatory response may have pathological consequences, since inflammation is a major defensive mechanism and its efficiency maintains the body’s health. We can thus consider as pathology-related inflammation those inflammatory reactions that, instead of eliminating foreign agents, lack down-regulation and cause tissue damage. To assess the ability of nanoparticles to initiate and modulate inflammatory reactions, an in vitro model was used that recapitulates all the stages of infection-induced inflammation, from initiation to resolution, based on human primary blood monoytes. A parallel model reproducing pathological chronic inflammation shows that the differences between resolving and persistent inflammation are subtle and evident only upon kinetic analysis of gene expression profiles and production of inflammatory factors. Rigorously endotoxin-free Au and Ag nanoparticles have been assessed for their ability to directly initiate in vitro inflammation and for their capacity to modulate the course both physiological resolving inflammation and pathological persistent inflammation. In no case significant effects were observed, with the exception of a transient increase of the inflammatory response in the presence of Ag nanoparticles. An important issue in the regulation of monocyte/macrophage inflammatory functions is the capacity of innate “memory”, i.e., the ability of respond differently to a challenge if previously primed with the same or a different agent. How nanoparticles can impact innate memory was assessed by using Au nanoparticles as priming and challenge agent with and without LPS and zymosan. Priming with LPS and zymosan could drastically decrease the response of monocytes (production of TNFa) to a challenge with any stimulus, given 7 days after the first. The presence of Au nanoparticles did not influence such behaviour. Likewise, Au nanoparticles did not directly induce memory, i.e., did not influence the response of monocytes to subsequent stimuli. We conclude that Au and Ag nanoparticles, at the size and concentrations used, are taken up by monocytes without this causing any notable interference with their capacity to mount an adequate defensive responses to microbial challenges, either immediate or after some time from exposure. This work was supported by per EU FP7 projects HUMUNITY and BioCog, the H2020 project PANDORA, the CNR Flagship Project InterOmics, and the cluster project Medintech of the Italian Ministry of Education, University and Research

The Interactions between Nanoparticles and the Innate Immune System from a Nanotechnologist Perspective

Inflamació; Immunitat innata; NanopartículesInflamación; Inmunidad innata; NanopartículasInflammation; Innate immunity; NanoparticlesThe immune system contributes to maintaining the body’s functional integrity through its two main functions: recognizing and destroying foreign external agents (invading microorganisms) and identifying and eliminating senescent cells and damaged or abnormal endogenous entities (such as cellular debris or misfolded/degraded proteins). Accordingly, the immune system can detect molecular and cellular structures with a spatial resolution of a few nm, which allows for detecting molecular patterns expressed in a great variety of pathogens, including viral and bacterial proteins and bacterial nucleic acid sequences. Such patterns are also expressed in abnormal cells. In this context, it is expected that nanostructured materials in the size range of proteins, protein aggregates, and viruses with different molecular coatings can engage in a sophisticated interaction with the immune system. Nanoparticles can be recognized or passed undetected by the immune system. Once detected, they can be tolerated or induce defensive (inflammatory) or anti-inflammatory responses. This paper describes the different modes of interaction between nanoparticles, especially inorganic nanoparticles, and the immune system, especially the innate immune system. This perspective should help to propose a set of selection rules for nanosafety-by-design and medical nanoparticle design.This research was funded by the EU Commission H2020 project PANDORA (GA 671881; to D.B., P.I. and V.P.). Additional funds were provided by the EU Commission H2020 project ENDONANO (GA 812661; to P.I. and D.B.), the Italian MIUR InterOmics Flagship projects MEMORAT and MAME (to D.B. and P.I.), the Italian MIUR/PRIN-20173ZECCM (to P.I.), the CAS President’s International Fellowship Programme (PIFI; award 2020VBA0028; to D.B.), Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU) (RTI2018-099965-B-I00, AEI/FEDER, UE), and Generalitat de Catalunya (2017-SGR-1431) (V.P.)

Profiling the Course of Resolving vs. Persistent Inflammation in Human Monocytes: The Role of IL-1 Family Molecules

Monocytes and macrophages have a central role in all phases of an inflammatory reaction. To understanding the regulation of monocyte activation during a physiological or pathological inflammation, we propose two in vitro models that recapitulate the different phases of the reaction (recruitment, initiation, development, and resolution vs. persistence of inflammation), based on human primary blood monocytes exposed to sequential modifications of microenvironmental conditions. These models exclusively describe the functional development of blood-derived monocytes that first enter an inflammatory site. All reaction phases were profiled by RNA-Seq, and the two models were validated by studying the modulation of IL-1 family members. Genes were differentially modulated, and distinct clusters were identified during the various phases of inflammation. Pathway analysis revealed that both models were enriched in pathways involved in innate immune activation. We observe that monocytes acquire an M1-like profile during early inflammation, and switch to a deactivated M2-like profile during both the resolving and persistent phases. However, during persistent inflammation they partially maintain an M1 profile, although they lose the ability to produce inflammatory cytokines compared to M1 cells. The production of IL-1 family molecules by ELISA reflected the transcriptomic profiles in the distinct phases of the two inflammatory reactions. Based on the results, we hypothesize that persistence of inflammatory stimuli cannot maintain the M1 activated phenotype of incoming monocytes for long, suggesting that the persistent presence of M1 cells and effects in a chronically inflamed tissue is mainly due to activation of newly incoming cells. Moreover, being IL-1 family molecules mainly expressed and secreted by monocytes during the early stages of the inflammatory response (within 4-14 h), and the rate of their production decreasing during the late phase of both resolving and persistent inflammation, we suppose that IL-1 factors are key regulators of the acute defensive innate inflammatory reaction that precedes establishment of longer-term adaptive immunity, and are mainly related to the presence of recently recruited blood monocytes. The well-described role of IL-1 family cytokines and receptors in chronic inflammation is therefore most likely dependent on the continuous influx of blood monocytes into a chronically inflamed site

Methodological Approaches To Assess Innate Immunity and Innate Memory in Marine Invertebrates and Humans

open8Assessing the impact of drugs and contaminants on immune responses requires methodological approaches able to represent real-life conditions and predict long-term effects. Innate immunity/inflammation is the evolutionarily most widespread and conserved defensive mechanism in living organisms, and therefore we will focus here on immunotoxicological methods that specifically target such processes. By exploiting the conserved mechanisms of innate immunity, we have examined the most representative immunotoxicity methodological approaches across living species, to identify common features and human proxy models/assays. Three marine invertebrate organisms are examined in comparison with humans, i.e., bivalve molluscs, tunicates and sea urchins. In vivo and in vitro approaches are compared, highlighting common mechanisms and species-specific endpoints, to be applied in predictive human and environmental immunotoxicity assessment. Emphasis is given to the 3R principle of Replacement, Refinement and Reduction of Animals in Research and to the application of the ARRIVE guidelines on reporting animal research, in order to strengthen the quality and usability of immunotoxicology research data.openAuguste, Manon; Melillo, Daniela; Corteggio, Annunziata; Marino, Rita; Canesi, Laura; Pinsino, Annalisa; Italiani, Paola; Boraschi, DianaAuguste, Manon; Melillo, Daniela; Corteggio, Annunziata; Marino, Rita; Canesi, Laura; Pinsino, Annalisa; Italiani, Paola; Boraschi, Dian

Different Regulation of Interleukin-1 Production and Activity in Monocytes and Macrophages: Innate Memory as an Endogenous Mechanism of IL-1 Inhibition

Production and activity of interleukin (IL)-1β are kept under strict control in our body, because of its powerful inflammation-promoting capacity. Control of IL-1β production and activity allows IL-1 to exert its defensive activities without causing extensive tissue damage. Monocytes are the major producers of IL-1β during inflammation, but they are also able to produce significant amounts of IL-1 inhibitors such as IL-1Ra and the soluble form of the decoy receptor IL-1R2, in an auto-regulatory feedback loop. Here, we investigated how innate immune memory could modulate production and activity of IL-1β by human primary monocytes and monocyte-derived tissue-like/deactivated macrophages in vitro. Cells were exposed to Gram-negative (Escherichia coli) and Gram-positive (Lactobacillus acidophilus) bacteria for 24 h, then allowed to rest, and then re-challenged with the same stimuli. The presence of biologically active IL-1β in cell supernatants was calculated as the ratio between free IL-1β (i.e., the cytokine that is not bound/inhibited by sIL-1R2) and its receptor antagonist IL-1Ra. As expected, we observed that the responsiveness of tissue-like/deactivated macrophages to bacterial stimuli was lower than that of monocytes. After resting and re-stimulation, a memory effect was evident for the production of inflammatory cytokines, whereas production of alarm signals (chemokines) was minimally affected. We observed a high variability in the innate memory response among individual donors. This is expected since innate memory largely depends on the previous history of exposure or infections, which is different in different subjects. Overall, innate memory appeared to limit the amount of active IL-1β produced by macrophages in response to a bacterial challenge, while enhancing the responsiveness of monocytes. The functional re-programming of mononuclear phagocytes through modulation of innate memory may provide innovative approaches in the management of inflammatory diseases, as well as in the design of new immunization strategies. In this respect, the interindividual variability in innate memory suggests the need of a personalized assessment