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Digital archive of theses discussed at the University of Pisa


Thesis etd-08082013-142015

Thesis type
Tesi di dottorato di ricerca
Thesis title
An in vitro kinetical model of human monocyte reactivity during a prototypical viral inflammation
Academic discipline
Course of study
tutor Prof. Migliorini, Paola
tutor Dott. Boraschi, Diana
  • cytokine
  • Inflammation
  • monocyte
  • virus
Graduation session start date
The innate/inflammatory defensive reaction is activated in response to invading or stressful events of different origin: viruses, bacteria, stress factors, tumour cells. Inflammation is an extremely complex reaction that involves many different actors, interacting with each other and with environmental factors. This complexity is an obstacle to the understanding of the role and function of individual elements, so that a simplification of the system is necessary if we want to focus on the specific behaviour of a particular element, like monocyte during viral inflammatory reaction. Monocytes/macrophages are among the first cells that are recruited to the site of inflammation and trigger the defence mechanisms by initiating different activation programmes. The aim of this work is to set up an in vitro model based on human monocytes that simulates the key steps of the inflammatory reaction to a viral challenge, from pathogen-induced triggering to eventual resolution and re-establishment of tissue homeostasis.
CD14+ monocytes are isolated from buffy coats of healthy donors and exposed sequentially in culture to the chemokine CCL2 (from 0 to 2 h, 37°C), viral inflammatory stimuli, i.e. a mixture of synthetic polyuridines, CpG motifs and polyI:C (from 2 to 14 h, 39°C), IFN-β/α (from 3 to 14 h, 39°C), IFN-γ (from 7 to 14 h, 39°C), IL-10 (from 14 to 24 h, 37°C), and TGF-β (from 24 h, 37°C). Samples of cells and supernatants were collected at different time points and the expression and the protein production pattern of some inflammatory cytokines, their inhibitors and chemokines were analyzed. Results from our study show that during the inflammatory phase, from 2 to 14 h, there is a significant increase of the production of IL-1β, free IL-18, IL-6 and TNF-α by monocytes, while they are down-regulated in the resolution and tissue repair phases. These data demonstrates the ability of the viral stimuli used in our model to trigger an inflammatory reaction, and it is also evident how the inflammatory reaction can be controlled by the induction of cytokine inhibitors and the down-regulation of the inflammatory mediators. Ultimately, these events allow the inhibition of inflammation and reestablishment of homeostasis.
On the basis of these results we can conclude that the monocytes-based in vitro model of development and resolution of inflammation seems able to reproduce in a reliable and representative fashion the human innate/inflammatory response to a viral challenge. Our model could find an effective application in defining the profile of the physiological response to specific viruses, as different viral agents could have different effects on an inflammatory reaction.
By comparing the viral model to bacterial-induced inflammation in a parallel model, we found that both the stimulations promote the release of similar set of cytokines and chemokines by monocytes/macrophages, suggesting that a classical activation of monocyte can be induced both by bacteria and viruses.
This monocyte-based model could be used to identify molecular signatures characteristic of the various phases of the inflammatory process in different situations, such as physiological vs. pathological conditions. Moreover, it could be used to study the behaviour of immune cells in different conditions, like co-infection, chronic inflammation, or under the effects of anti-inflammatory drugs or nanoparticles.