• innate immunity
• inflammation
• chemokine
• macrophage
• IL-18
• monocyte
• monocyte polarization
• cytokine
• IL-1R
• IL-1

Abstract
To start a successful infection, pathogens need to cross the physical barriers lining our body, the epithelia (skin; respiratory, gastrointestinal, urogenital mocosae). However, epithelia are not inert physical barriers, and within the epithelia the pathogen will encounter the first active defense system of the host. Among the cells of the immune system patrolling epithelial linings, an important role is represented by resident macrophages. Upon contact with different types of pathogens, macrophages can undergo a polarised pattern of activation and start secreting various factors to recruit and activate other cells of the immune system such as blood monocytes, neutrophils, dendritic cells, natural killer cells and lymphocytes. The innate immune system is most of the times able to block an infection without the need of an adaptative response. Innate immune cell receptors can recognise moleculear patterns characteristic of microbes and become activated to directly eliminate the invading pathogen. If an adaptative response is necessary, it is the innate immune system that triggers this activation: cells of the innate immunity present the antigen to lymphocytes and keep the pathogen in the infection site while the organism sets up the adaptative response.
Cells of the innate immune system have different roles during a pathogenic infection. Neutrophils are specialised phagocytic cells that usually die during the infection. Dendritic cells leave the site of inflammation after uptake of pathogen and reach the lymph nodes where they present the antigen to lymphocytes. Resident macrophages are present in the site of infection and are the first to come in contact with the pathogens. Activated macrophages secrete bactericidal molecules, can directly engulf and destroy pathogens and initiate the inflammatory reaction that limits and stops the infection. Indeed, pathogen-activated macrophages produce chemokines that attract to the site of infection inflammatory leukocytes from the blood (PMN, monocytes) and cytokines that activate the recruited leukocytes. When the infection is stopped and pathogens destroyed, the inflammatory activation of macrophages and recruited monocytes in the reaction site stops, and cells react to the microenvironmental change by producing factors that promote tissue repair, cell proliferation and angiogenesis in the damaged tissue, thus re-establishing tissue homeostasis.
Blood monocytes recruited to the site of infection can initiate two different types of activation programmes depending on the characteristics of the infectious/inflamatory event.Classical inflammatory cytocidal activation ( M1 polarisation) is triggered by recognition of bacterial or viral pathogen-associated molecular patterns (PAMPs) and amplified in the presence of the pro-inflammatory cytokine IFN-gamma produced by NK cells and T lymphocytes. M1 macrophages are potent producers of inflammatory factors (e.g., the cytokines IL-1beta, IL-12, TNF-alpha) and chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, etc.), reactive oxygen species and nitrogen monoxide. M1 polarisation increases the phagocytic activity of macrophages and phagosome acidification, increasing ingestion and destruction of pathogens. On the other hand, the M2 polarisation is induced by different types of pathogens/stimuli (e.g. helmints) and it can be defined as a “non-M1”. M2 macrophages have diverse characteristics but have in common low production of IL-12 and IL-23 and high production of IL-10. At least three different types of M2 polarisation have been described. One is the alternative inflammatory activation (very different from M1) that is initiated in response to invasion by worms and parasites, characterised by the production of alternative inflammatory factors (such as IL-4) in parallel to the production of factors that down-regulate classical inflammation (such as IL-10 and IL-1Ra). Another one is a non-classical inflammatory activation with concomitant production of classical inflammatory cytokines and anti-inflammatory factors, while a last group of alternatively activated M2 types can be considered as “de-activated” M2 polarisation and generally include lower production of classical inflammatory molecules, and concomitant promotion of angiogenesis and tissue remodelling.
The aim of this work is to build and validate an in vitro model system that simulates the key steps of the inflammatory reaction, from pathogen-induced triggering to eventual resolution and re-establishment of homeostasis. The system is based on human blood monocytes and on their M1 and M2 polarisation in response to prototypical stimuli simulating the conditions of the tissue microenvironment during the various phases of the inflammatory process. During the in vitro reaction, samples of cells and supernatants will be taken at different time points, to be analysed with proteomics and transcriptomics platforms. Particolar attention will be devoted to the study of the cytokines and receptors of the IL-1 family.
IL-1-like cytokines include eleven cytokines involved both in the inflammatory and anti-inflammatory responses. It is known that defects in the regulation of IL-1 cytokine production/activity correlate with the development of chronic inflammatory and autoimmune diseases such as rheumatoid arthritis, Crhon’s disease, multiple sclerosis, and systemic lupus erythematosus.
While high-throughput transcriptomics and proteomics studies (by microarrays and mass spectrometry) are still underway, in this work we present preliminary data aimed at confirming the robustness of the in vitro model system and its representativity of the inflamamtory reaction. Moreover we present the first data describing the modulation of the of IL-1/IL-1R molecules in macrophages during all phases of inflammation in our model system.