Understanding the Collective Dynamics of Motile Cilia in Human Airways

Eukaryotic organisms rely on the coordinated beating of motile cilia for a multitude of fundamental reasons. In smaller organisms, such as Paramecium and the single cell alga Chlamydomonas reinhardtii, it is a matter of propulsion, to swim towards a higher concentration of nutrients or away from damaging environments. Larger organisms use instead the coordinated motion of cilia to push fluid along an epithelium: examples common to mammals are the circulation of cerebrospinal fluid in the brain, the transport of ovules in the fallopian tubes, and breaking the left/right symmetry in the embryo. Another notable example, and one that is central to this thesis, is mucociliary clearance in human airways: A carpet of motile cilia helps keeping the cell surface free from pathogens and foreign particles by constantly evacuating from lungs, bronchi, and trachea a barrier of mucus. The question of how motile cilia interact with one another to beat in a coordinated fashion is an open and pressing one, with immediate implications for the medical community. In order for the fluid propulsion to be effective, the motion of cilia needs to be phase-locked across significant distances, in the form of travelling waves (``metachronal waves''). It is still not known how this long-range coordination emerges from local rules, as there is no central node regulating the coordination among cilia. In the first part of this thesis I will focus on studying the coordination in carpets of cilia with a top-down approach, by proposing, implementing, and applying a new method of analysing microscope videos of ciliated epithelia. Chapter 1 provides the reader with an introduction on motile cilia and flagella, treating their structure and motion and reporting the different open questions currently tackled by the scientific community, with particular interest in the coordination mechanisms of cilia and the mucociliary clearance apparatus. Chapter 2 introduces Differential Dynamic Microscopy (DDM), a powerful and versatile image analysis tool that bridges the gap between spectroscopy and microscopy by allowing to perform scattering experiments on a microscope. The most interesting aspects of DDM for this work are that it can be applied to microscope videos where it is not possible to resolve individual objects in the field of view, and it requires no user input. These two characteristics make DDM a perfect candidate for analysing several hundred microscope videos of weakly scattering filaments such as cilia. In Chapter 3 I will present how it is possible to employ DDM to extract a wealth of often-overlooked information from videos of ciliated epithelia: DDM can successfully probe the ciliary beat frequency (CBF) in a sample, measure the direction of beating of the cilia, and detect metachronal waves and read their direction and wavelength. In vitro ciliated epithelia however often do not show perfect coordination or alignment among cilia. For the analysis of these samples, where the metachronal coordination might not be evident, we developed a new approach, called multiscale DDM (multiDDM), to measure a coordination length scale, a characteristic length of the system over which the coordination between cilia is lost. The new technique of multiDDM is employed in Chapter 4 to study how the coordination among cilia changes as a response to changes in the rheology of the mucous layer. In particular, we show that cilia beating under a thick, gel-like mucus layer show a larger coordination length scale, as if the mucus acted as an elastic raft effectively coupling cilia over long distances. This is corroborated by the coordination length scale being larger in samples from patients affected by Cystic Fibrosis than in healthy samples, and much shorter when the mucus layer is washed and cilia therefore beat in a near-Newtonian fluid. We then show how it is possible to employ multiDDM to measure the effectiveness of drugs in recovering, in CF samples, a coordination length scale typical of a healthy phenotype. In the second part I will focus instead on the single cilium scale, showing how we can attempt to link the beating pattern of cilia to numerical simulations studying synchronisation in a model system. In particular in Chapter 5 I will describe our approach to quantitatively describe the beating pattern of single cilia obtained from human airway cells of either healthy individuals or patients affected by Primary Ciliary Dyskinesia. Our description of the beating pattern, and the selection of a few meaningful, summary parameters, are then shown to be accurate enough to discriminate between different mutations within Primary Ciliary Dyskinesia. In Chapter 6 instead I report the results obtained by coarse-graining the ciliary beat pattern into a model system consisting of two ``rotors''. The rotors are simulated colloidal particles driven along closed trajectories while leaving their phase free. In my study, the trajectories followed by the rotors are analytical fits of experimental trajectories of the centre of drag of real cilia. The rotors, that are coupled only via hydrodynamics interactions, are seen to phase-lock, and the shape of the trajectory they are driven along is seen to influence the steady state of the system

Development of optical Doppler interferometry for the visualization of ocular elasticity and ciliary activity

Functional optical imaging techniques have become increasingly important due to their high resolution and non-invasive nature, and have been used to address many unmet needs in the biomedical imaging field. In the area of ophthalmology, mechanical properties have been shown to be an early indicator of retinal disease, but current imaging modalities are unable to provide high resolution in-vivo imaging to capture the minute changes in the elasticity of thin tissue layers at the back of the eye. For respiratory diseases, the ciliary cell function inside the airway have been discovered to play an important role in respiratory health and the onset of disease. Similarly, current techniques are not equipped to image and characterize the cellular level changes in in-vivo tissues. Phase-resolved Doppler (PRD) imaging is a technology developed by our F-OCT lab, primarily for visualizing blood flow and angiography. Recently, it has been determined that the PRD technique is able to provide high phase sensitivity, which can be used to obtain the tissue displacement as well as particle motions. Using this principle, we developed two types of imaging systems: confocal acoustic radiation force optical coherence elastography (ARF-OCE) and spectrally-encoded interferometric microscopy (SEIM). Using the confocal ARF-OCE system, we present the first spatially mapped elasticity imaging in a live animal retina, and obtained a better understanding of the elasticity of different retinal layers. With the SEIM system, we introduced a novel method of spatially tracking ciliary activity in real-time of in vitro tracheal and oviduct tissues. We demonstrate that the SEIM system can image and quantify ciliary beating frequency and ciliary beating pattern with high speed and large field of view. While both these technologies use the PRD technique, the optical system has been optimized for the respective applications. The results in this dissertation serve as a stepping stone to the optimization and ultimately, the clinical translation of the PRD technique to diagnostic imaging. The developed technology has great potential for clinical diagnosis and management of a number of ocular disease, such as age related macular degeneration, glaucoma, presbyopia and myopia, as well as airway diseases such as asthma

The interaction of bacteria with the respiratory mucosa in vitro and in vivo

Using a simple nasopharyngeal organ culture in which the mucosa is exposed to air, this thesis describes the interaction between two piliated and one non-piliated variants of Neisseria meningitidis and the interaction of a pneumolysin sufficient and deficient isogenic variant of Streptococcus pneumoniae with respiratory mucosa. Piliated N. meningitidis adhered more often than the non-piliated variant to the respiratory mucosa and demonstrated tropism for non-ciliated epithelial cells and only rarely adhered to mucus. In contrast, S.pneumoniae demonstrated tropism for mucus. Infection resulted in a change in the appearance of mucus, ciliary beat slowing and epithelial damage. To assess if other bacteria may impair mucociliary clearance by disorganising cilia the effect of pyocyanin, 1-hydroxyphenazine (1-HP) and rhamnolipid on the orientation of human ciliated cells was studied. Pyocyanin and 1-HP at pathophysiological concentrations caused ciliary slowing, dyskinesia and disorientation of the ciliary microtubular pairs. However, the orientation of basal feet did not change. Rhamnolipid at pathophysiologic concentrations caused ciliary slowing but neither dyskinesia or disorientation. Disorientation of ciliary beat as well as slowed CBF may contribute to the slowing of mucociliary clearance in vivo. To assess if ciliary disorientation occurs as an acquired and/or congenital abnormality, groups of patients with chronic upper respiratory tract inflammation due to infection and patients with the clinical features of primary ciliary dyskinesia but normal ciliary beat frequency and ciliary ultrastructure were studied. Ciliary disorientation was associated with slowing of nasomucociliary clearance. The clinical features, ciliary function studies and the ciliary orientation of eleven patients with the classical features of primary ciliary dyskinesia but with normal ciliary ultrastructure were assessed. The results suggests that ciliary disorientation alone does represent a new variant of primary ciliary dyskinesia

Novel Gene Discovery in Primary Ciliary Dyskinesia

Primary Ciliary Dyskinesia (PCD) is one of the ‘ciliopathies’, genetic disorders affecting either cilia structure or function. PCD is a rare recessive disease caused by defective motile cilia. Affected individuals manifest with neonatal respiratory distress, chronic wet cough, upper respiratory tract problems, progressive lung disease resulting in bronchiectasis, laterality problems including heart defects and adult infertility. Early diagnosis and management are essential for better respiratory disease prognosis. PCD is a highly genetically heterogeneous disorder with causal mutations identified in 36 genes that account for the disease in about 70% of PCD cases, suggesting that additional genes remain to be discovered. Targeted next generation sequencing was used for genetic screening of a cohort of patients with confirmed or suggestive PCD diagnosis. The use of multi-gene panel sequencing yielded a high diagnostic output (> 70%) with mutations identified in known PCD genes. Over half of these mutations were novel alleles, expanding the mutation spectrum in PCD genes. The inclusion of patients from various ethnic backgrounds revealed a striking impact of ethnicity on the composition of disease alleles uncovering a significant genetic stratification of PCD in different populations. Pathogenic mutations were also identified in several new candidate genes not previously linked to PCD. Molecular and cell biology techniques were coupled with model organism studies to characterize the involvement of the new candidate genes in cilia motility and PCD. Paramecium was proven to be a good model for functional characterization of PCD potential candidate genes. The previously uncharacterized C11orf70 was identified to play a highly conserved role in dynein assembly and intraflagellar transport (IFT)-related cilia cargo trafficking. Mutations identified in DNAH9 resulted in a distinct motile cilia defect with mild respiratory symptoms, unusual in PCD. Mutations identified in two intraflagellar transport genes, IFT74 and WDR19, linked together primary and motile ciliopathy phenotypes observed in the affected individuals

The role of hydrodynamic forces in synchronisation and alignment of mammalian motile cilia

Fluid flow generated by a ciliated epithelium is a fascinating evidence of collective behaviour in nature. In many organs and eukaryotic organisms, thousands of microscale whip-like structures called `motile cilia' beat aligned at the same frequency and in a coordinated fashion. This dynamics, known as `metachronal wave', has fundamental physiological roles in microorganisms and many organs of vertebrates. In the airways, the coordinated beatings of motile cilia generate a fluid flow that pushes mucus to the pharynx, and so protects the lungs from inhaled contaminants. The failure of this collective dynamics can precipitate or exacerbate severe infections and chronic inflammatory conditions such as cystic fibrosis (CF), primary ciliary dyskinesia (PCD) or asthma. In the brain, the multiciliated ependymal cells cover all the ventricles. Their cilia beat in a coordinated fashion to ensure the cerebrospinal fluid circulation necessary for brain homoeostasis, toxin washout and orientation of the migration of newborn neurons. Despite the fundamental role in nature, the mechanism underpinning such collective behaviour is still unknown. A recent hypothesis, supported by simulations, experiments with microorganisms and with cilia models, proposed that hydrodynamic interactions between cilia could provide a physical mechanism for their coordination. In contrast, others have proposed a role of the cytoskeletal elastic coupling between cilia. While previous works mainly focused on algae and protists, investigating the conditions that are required for the emergence of the metachronal wave in mammalian tissues can provide important progress in the diagnosis and treatment of human medical diseases. Specifically, I tackled this broad topic by studying the hydrodynamic forces necessary for the synchronisation and alignment of motile cilia from brain and airways. This question was addressed experimentally by measuring cilia motility during treatment with oscillatory and constant external fluid flows. We found that synchronisation and alignment of mammalian cilia in the brain is achieved with flows of similar magnitude of the ones generated by cilia themselves. Our results suggest that hydrodynamic forces between cilia are sufficient for the emergence of their collective behaviour. The first chapter provides basic knowledge on motile cilia structure and functions in microorganisms and humans. Additionally, I introduce the reader to the open questions related to the coordination of a pair and a carpet of cilia, with specific attention on previous works on mammals. This first chapter is followed by a description of a novel microfluidic device that I developed to grow $\textit{in vitro}$ airway and brain cells and apply controlled viscous forces. In Chapter 3, I describe how we have investigated cilia synchronisation of mammalian cilia. Applying external oscillatory flow on brain cells, we studied the susceptibility of cilia motility to hydrodynamic forces similar to the ones generated by cilia themselves. We found that cells with few cilia (up to five) can be entrained at flows comparable to the cilia-driven flows reported in vivo. We suggest that hydrodynamic forces between mammalian cilia are sufficiently strong to be the mechanism underpinning frequency synchronisation. In the second part of my thesis, I looked into the hydrodynamic shear forces needed to align permanently the cilia direction of beating. We tackled this problem by using $\textit{in vitro}$ cultures of mouse brain and human airway cells grown in custom flow channels. We found that cilia from mouse brain do not lock their beating direction after \emph{ciliogenesis}, but can respond and align to physiological shear stress found \emph{in vivo} at any time, in contrast with was previously believed. Moreover, we suggest that cilia alignment depends on the density of cilia, in agreement with a hydrodynamic screening effect of the external flow by the nearby cilia that we aim to investigate in the future. These results are described in Chapter 4. Successively in Chapter 5, I report our approach to study whether physiological shear stress can induce cilia alignment in airway cell cultures. The current hypothesis is that these cilia may also be able to align with external hydrodynamic forces - however, experimental evidence is still needed. There is a lack of experiments on this topic mainly because airway cells are cultured in an air-liquid interface, and so shear stress has to be applied with airflows. We developed novel setups for applying long term shear stress with air and fluid flow on this system, leaving further experiments for the future.EU Horizon 2020 research and innovation program under Marie Sklodowska-Curie 641639 ITN BioPol and ERC CoG HydroSyn

Rare Respiratory Diseases: A Personal and a Public Health Problem

Dear Colleagues, A rare disease, also known as an orphan disease, is any disease that affects a small percentage of the population. Although definitions vary from continent to continent, according to the European Union, rare diseases are those with a prevalence of less than 1 in 2000 people. Rare diseases are, in general, chronic, debilitating diseases, which in many cases threaten patients’ lives. It is estimated that 1–2 million people in the European Union are affected by a rare respiratory disease, which is a public health problem. Due to the low prevalence and severity of many of these diseases, whose symptoms often initially manifest in childhood, combined efforts are needed to improve our knowledge of the pathophysiology of these diseases that will lead to the development of new, more effective treatments. Therefore, since rare respiratory diseases represent an important field in medicine, we propose this Special Issue to promote the dissemination of the latest advances in basic and clinical research in these diseases. Prof. Dr. Francisco Dasí Guest Edito

Non-typeable Haemophilus influenzae and rhinovirus co-infection of the respiratory epithelium

Non-typeable Haemophilus influenzae (NTHi) and rhinovirus 16 (RV16) are strongly associated with symptomatic disease in healthy individuals and with disease exacerbations in patients with chronic lung conditions such as chronic obstructive pulmonary disease (COPD). Co-infection with RV16 and NTHi is thought to cause more severe exacerbations, although the mechanism behind this remains unknown. The hypothesis of this project was that RV16 and NTHi co-infection of the respiratory epithelium results in increased bacterial growth, greater epithelial damage and inflammation compared to single NTHi infection. To investigate this, healthy and COPD primary respiratory epithelial cells cultured at air-liquid interface for 6 days (pre-ciliation) or 28 days (ciliated) were infected with RV16 and 24 hours later challenged with NTHi. // In healthy and COPD ciliated epithelial cultures, NTHi bound to motile cilia within minutes, then formed filamentous morphotypes and biofilm-like aggregates which reduced ciliary beat amplitude by 24 hours post infection. NTHi epithelial invasion occurred preferentially in non-ciliated epithelial cells suggesting that ciliation may protect against NTHi invasion. This might be of importance in COPD cultures, where the number of ciliated cells was reduced, reflecting what is seen in vivo. // Co-infection of ciliated cultures with RV16 and NTHi resulted in a reduced ciliary beat frequency, epithelial barrier dysfunction and a more complex inflammatory response from healthy and COPD ciliated cultures, compared to NTHi single infection. RV16 co-infection also promoted mucin gene expression and enhanced NTHi growth by altering epithelial cell apical fluid secretion. In contrast, pre-ciliation cultures showed markedly reduced responses to single RV16 or NTHi infection and their co-infection compared to ciliated cultures. However, pre-ciliation cultures were susceptible to NTHi invasion and promoted NTHi growth during co-infection with RV16, suggesting they may act like a niche for bacterial colonisation. // In conclusion, this study has shown that NTHi infection is able to affect ciliary function, form biofilm and invade the epithelium without epithelial barrier damage or induction of a complex inflammatory response, suggestive of a colonizer behaviour. In contrast, co-infection with RV16 and NTHi led to NTHi growth, a goblet cell-specific transcriptional profile, impaired ciliary function and epithelial barrier and increased inflammation which could result in increased bacterial dissemination and COPD exacerbation

Diagnostic tools in Rhinology EAACI position paper

This EAACI Task Force document aims at providing the readers with a comprehensive and complete overview of the currently available tools for diagnosis of nasal and sino-nasal disease. We have tried to logically order the different important issues related to history taking, clinical examination and additional investigative tools for evaluation of the severity of sinonasal disease into a consensus document. A panel of European experts in the field of Rhinology has contributed to this consensus document on Diagnostic Tools in Rhinology

Primary ciliary dyskinesia: a biopsychosocial approach

Background: Primary ciliary dyskinesia (PCD) is a rare heterogeneous genetic disorder associated with abnormal ciliary structure and function and characterised by progressive sinopulmonary disease. There is no ‘gold standard’ for diagnosing PCD. This thesis aimed: to provide an overview of the PCD patient’s experience, to address some of the complexities in referring and diagnosing PCD patients, and to provide PCD-specific validated health-related quality of life (HRQoL) measures. Method: A patient survey to capture patient experience of diagnostic testing was completed in 25 countries and was followed by semi-structured interviews, analysed thematically. Through the analysis of data from consecutive patients referred to a PCD diagnostic centre (2007-2013), sensitivity and specificity were calculated. Patient clinical characteristics were correlated with diagnostic outcome. Using logistic regression, the predictive performance of the best model was simplified into a tool (PICADAR), predicting the likelihood of referrals having PCD. A PCD-specific HRQoL measure (QOL-PCD) was developed following a literature review, expert panel meeting, and semi-structured interviews (n=21). Validation followed with patients completing QOL-PCD and generic HRQoL measures. Results: PCD was found to have a negative impact on physical, emotional, and social functioning, highlighting need for PCD-specific HRQoL measures. Outcome data for diagnostic testing showed that none of the diagnostic testing strategies were 100% accurate. PICADAR has been shown to have good accuracy and validity to predict diagnostic outcome. The QOL-PCD HRQoL demonstrated good internal consistency, test-retest reliability, convergent and divergent validity. Discussion: Findings from the international survey were used to advise on ERS Task Force guidelines for diagnosing PCD. The development of PICADAR will lead to earlier referral of patients. The development of QOL-PCD provides the first disease-specific measure to allow for the assessment of treatments. Overall this thesis has led to advances in the field of PCD; from diagnosis to the treatment and management of patients