Objective
Assessing the mucosal innate/inflammatory reactivity to prototypical inhaled challenges (bacteria, allergens, particles) in a cellular model of the tracheal/upper respiratory tract mucosa.

Abstract
The mucosal layer lining the upper respiratory tract plays a vital role in preventing infection and allergic reactions. This mucosal lining is one of the body’s first lines of defence against airborne microbes and other harmful agents. Immune cells in the mucosa play a key role in this defence, and may provoke inflammation in response to repetitive exposure to aggressive agents.
The biology of upper respiratory tract response to potentially detrimental agents is not fully understood, and few laboratory models are capable of replacing experimental animals in studies to elucidate the interactions of the cells and molecules involved. Moreover, there is a lack of biological models for the development of new therapeutic interventions. Industry relies on potentially imprecise models that use animals, or which assemble human mucosal components in ways that do not reflect the body’s true architecture or physiology, thus diminishing the reliability and predictive power of tests.
The main purpose of this project is to develop human in vitro models of the trachea by adapting innovative cell-based technologies, and ensuring reproducibility and precise functioning of biological processes that occur during the course of inflammation. The project will focus on isolating, characterising and re-assembling the mucosal cells from the upper airways of healthy and ill donors. The possibility of sourcing such human tissue samples under full ethical consent will allow the development of predictive models of chronically inflamed airways (e.g. chronic obstructive pulmonary disease). Advanced biomaterials will assemble cells in a controlled manner, into physiologically-relevant architectures. Alongside this, the project shall also validate ways of measuring inflammatory events in terms of gene expression, enzymatic activity and cell-to-cell signalling.
The expected outcome of the project will be a well-characterised and validated panel of cell-based models capable of acting as a screening platform for drug discovery against life-threatening conditions of the upper respiratory tract. Such powerful human cell-based models may also be used to identify airborne agents that interfere with normal respiratory function.

Update
In the period from April to August 2016, James conducted a series of experiments intended to consolidate the ALI (air-liquid interface) culture of human primary lung epithelial cells. His experiments investigated the longevity of these cultures, and tested factors which could affect the integrity of monolayers formed by the cells on permeable membranes. The ultimate objective of his project has been to establish co-cultures of these human lung cell monolayers with sub-tending human immune cells derived from peripheral blood. To this end, immune cell cultures produced in accordance with in-house Standard Operating Procedures have been characterised under conditions adapted to co-culture and the creation of a lung mucosal immune system. Factors determining epithelial cell viability have been identified; further work will be required to determine cytokine responses subject to epithelial cell modulation. James left the European Industrial Doctorate project in August 2016.

Objective
Evaluating the innate/inflammatory responses of the human bronchiolar tissue in pathological situations of cystic fibrosis, by implementing in vitro cell-based models.

Abstract
The lining of the respiratory tract provides a crucial physicochemical barrier against inhaled microbes and air pollutants. The mucosal immune system of this airway epithelium has a key role in anti-microbial defence and in protection against foreign materials. However, impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to chronic lung disease characterised by persistent infections and prolonged inflammation.
Tissue cultures and three-dimensional cell cultures are used in biomedical research, because these advanced cell culture techniques are cost-effective compared with animal models, and provide a potential for high-throughput screening generating physiologically-relevant data. Moreover, the development of novel lung models has emerged as a promising tool to mimic the human lung mucosal immune system in order to reproduce the cellular mechanism involved in cystic fibrosis.
This project shall undertake research into and development of novel 3D cell models of lung mucosa, and shall specifically focus on isolating, characterising, handling, storing and re-assembling the lung mucosal cells which make up this ex vivo lung system. With this cell-based approach cellular models of healthy and cystic fibrotic epithelium will be set in place, to allow comparative testing of their response to challenge at the cellular and molecular levels. In order to study these systems, biochemical, immunological and gene expression techniques measuring their innate/inflammatory activation will be performed.
The project outcome is expected to be a well-characterised, validated cell-based model, which will be used to evaluate the interaction with, and efficacy of, novel therapeutics against cystic fibrosis.

Update
At CNR, Gergo has established the Air-Liquid Interface co-cultures (ALI culture) based on primary human bronchial epithelial cells and monocytes. The results show that the inflammatory reactivity in the co-culture model is mainly due to the presence of monocytes and the possible crosstalk between the various cell types. The established co-culture lung model reproduced the key characteristics of innate immune responses in an inflammatory environment. In September, he gave an oral presentation about his final in vitro co-culture lung model in the ‘International Retreat of PhD Students in Immunology’ coordinated by Società italiana di Immunologia, Immunologia Clinica e Allergologia in Naples.

Objective
Mapping at a cellular and molecular level the effects of the pathological condition on colonic innate/inflammatory defensive functions by reconstituting in vitro a model of the human colonic mucosa.

Abstract
The human body is constantly interacting with its environment. Skin, lung and, just as importantly, the intestine, play crucial roles in this interaction. Not only does the intestine absorb nutrients from the diet, but it is also responsible for the uptake of orally administered drugs and food constituents with bioactive as well as nutritional value. Moreover, as one of those outward-facing tissues, the gut serves as a line of defence against potentially harmful substances and organisms. To do so, it comprises several defence mechanisms, including a layer of mucus and the action of immune cells within the surface cell lining, called mucosa.
The scope of this project is to describe the cellular and molecular processes of the human intestinal mucosal immune system by means of a model system. So far, animal models are commonly used to investigate the relationship of agents presented at the mucosal surface and the reaction of the tissue to them. However, animal models often differ significantly from the situation in the human body. As alternatives, a limited number of cell based laboratory models are available. Unfortunately, these models typically do not include elements of the immune system, or do not assemble human mucosal components in a physiological-relevant manner, leading to unreliable and inaccurate results. To address this unmet need, the project aims to develop a new cell-based model of mucosal immunity in the large intestine, one which can be used as a more reliable tool when testing candidate therapeutics, health promoting agents or dietary contaminants for beneficial or harmful effects.
The culture system will be built from mucosal cells isolated from human tissue and characterised in terms of the cell sub-types resident within the tissue. The re-assembly of these cells into tissue-relevant architectures will be conducted by means of advanced biomaterials that serve as an artificial scaffold for the cells, and reconstitute the paracrine and autocrine signalling of the original tissue. This system shall be examined in terms of the cell and molecular mechanisms of inflammatory events, such as the release of signal molecules upon an inflammatory trigger.
The project aims to establish a validated cell-based model that robustly reproduces the reactivity of human large-intestinal mucosa. This preclinical model will offer multiple applications, such as screening therapeutics or dietary contaminants, reducing the need for animal experimentation.

Update
In the last stage of her project Elfi has been developing and characterizing a monolayer culture of differentiated colon epithelial cells derived from stem cell containing colonoids. This culture provides an attractive testing platform to assay epithelial responses to various stimuli. Furthermore, it has the potential to build a preclinical model system focused on innate immunity, if paired with immunological culture modules that Elfi has set up during the first part of the project.

Objective
To use personalised in vitro cell-based models of human pathological intestine for identifying the role of innate/inflammatory mechanisms and subsequently assess the effects of therapeutic (anti-inflammatory) agents active in inflammatory bowel disease (IBD).

Abstract
The project’s main objective is to develop in vitro models that mimic the architecture and functionality of the small intestine, the site of Crohn’s disease. This chronic inflammation is a form of inflammatory bowel disease (IBD), whose prevalence is increasing, especially in the industrialized countries of northern Europe and North America. Crohn’s disease can limit quality of life due to pain, vomiting, diarrhoea, and through non-gastrointestinal complications such as anaemia, arthritis or inflammation of the eyes.
Crohn’s disease inflammation affects the whole bowel wall and disrupts the balance between self-renewal and differentiation of columnar epithelial cells, a main component of the inner layer of the small intestine. Accordingly, the project’s models will be built with primary human intestinal epithelial cells isolated from healthy and pathological donors, which shall be cultured on advanced biomaterials alongside other cell types present in the original tissue. Organisation and functionality of the intestinal model system shall be evaluated by expression, maturation and trafficking of inflammatory cytokines and other key molecules involved in the inflammatory response, using a range of cellular, molecular and immunological techniques.
These new culture systems will deliver preclinical models for characterisation of the molecular mechanisms that define the inflamed tissue, up to and including the role of gut bacteria implicated in Crohn’s disease aetiology. These validated models shall be utilised for the evaluation and development of new therapeutic agents, reducing need for animal testing of IBD intervention.

Update
During the last months of the PhD project, Mariusz finalized an in vitro model of human inflamed small intestinal mucosa. The final model was built with generated primary human tissue-like macrophages and differentiated Caco-2 cells cultured on suitable membranes that allow direct cell-cell interactions. To reproduce an inflammatory condition, the epithelial monolayer was pre-treated with the inflammatory cytokine IL-1β. Then, the co-culture model was challenged apically with various intestinal bacteria and tested with an anti-inflammatory compound. Epithelial integrity, and production of inflammatory cytokines and chemokines were assessed in order to validate the final model. Data obtained suggest that the simplified in vitro co-culture model reproduces the main characteristics of innate immune response by tissue-resident macrophages during gut inflammation. The model thus is a good basis for developing commercially useful tissue-like assays for screening of drugs and contaminants

Moreover, Mariusz participated to the EFIS-EJI Advanced Course ‘Metchnikoff's legacy: tissue phagocytes and functions’, organised by the Ceppellini School, and to the ‘International Retreat of PhD Students in Immunology’, organised by the Italian Society of Immunology (SIICA) in Naples. During these meetings, Mariusz presented two posters and discussed his final PhD work and engaged with important immunologists. Currently, Mariusz is finalizing his PhD thesis and writing a manuscript on the regulation of cytokines and receptors of the IL-1 family in the context of innate immune memory.