Development of dendritic cells in the intestine

The intestinal tract is exposed to a large variety of antigens such as food proteins, commensal bacteria and pathogens and contains one of the largest arms of the immune system. The intestinal immune system has to discriminate between harmless and harmful antigens, inducing tolerance to harmless antigens and active immunity towards pathogens and other harmful materials. Dendritic cells (DC) in the mucosal lamina propria (LP) are central to this process, as they sample bacteria from the local environment and constitutively migrate to the draining mesenteric lymph nodes (MLN), where they present antigen to naïve T cells in order to direct an appropriate immune response. Despite their crucial role, understanding the function and phenotype of LP DC has been hampered by the fact that they share phenotypic markers with macrophages (mφ), which are the dominant population of mononuclear phagocyte (MP) in the LP. Recent work in our own and other laboratories has established gating strategies and phenotyping panels that allow precise discrimination between intestinal DC and mφ using the mφ specific markers CD64 and F4/80. In this way four bona fide DC subsets with distinct functions have been identified in adult LP based on their expression of CD11b and CD103 and a major aim of my project was to understand how these subsets might develop in the neonatal intestine. At the beginning of my PhD, the laboratory had used these new methods to show that signal regulatory protein α (SIRPα), an inhibitory receptor expressed by myeloid cells, was expressed by mφ and most DC in the intestine, except for those expressing CD103 alone. In addition, mice carrying a non-signalling mutation in SIRPα (SIRPα mt) had a selective reduction in CD103+CD11b+ DC, a subset which is unique to the intestinal LP. This was the basis for the initial experiments of my project, described in Chapter 3, where I investigated if the phenotype in SIRPα mt mice was intrinsic to haematopoietic cells or not. To explore this, I generated bone marrow (BM) chimeric mice by reconstituting irradiated WT mice with SIRPα mt BM, or SIRPα mt animals with WT BM. These experiments suggested that the defect in CD103+CD11b+ DC was not replicated in DC derived from BM of SIRPα origin. However as this seemed inconsistent with other data, I considered the possibility that 18 the phenotype may have been lost with age, as the BM chimeric mice were considerably older than those used in the original studies of SIRPα function. However a comparison of DC subsets in the intestine of WT and SIRPα mt mice as they aged provided no conclusive evidence to support this idea. As these experiments did show age-dependent effects on DC subsets, in Chapter 4, I went on to investigate how the DC populations appeared in the intestine and other tissues in the neonatal period. These experiments showed there were few CD103+CD11b+ DC present in the LP and migratory DC compartment of the MLN in the neonate and that as this population gradually increased in proportion with age, there was a reciprocal decrease in the relative proportion of CD103-CD11b+ DC. Interestingly, most of the changes in DC numbers in the intestine were found during the second or third week of life when the weaning process began. To validate my findings that there were few CD103+CD11b+ DC in the neonate and that this was not merely an absence of CD103 upregulation, I examined the expression of CD101 and Trem-1, markers that other work in the laboratory had suggested were specific to the CD103+CD11b+ DC lineage. My work showed that CD101 and Trem-1 were co- expressed by most CD103+CD11b+ DC in small intestine (SI) LP, as well as a small subset of CD103-CD11b+ DC in this tissue. Interestingly, Trem-1 was highly specific to the SI LP and migratory DC in the MLN, but absent from the colon and other tissues. CD101 expression was also only found on CD11b+ DC, but showed a less restricted pattern of distribution, being found in several tissues as well as the SI LP. The relative timing of their development suggested there might be a relationship between CD103+CD11b+ and CD103-CD11b+ DC and this was supported by microarray analysis. I hypothesised that the CD103-CD11b+ DC that co-expressed CD101 and Trem-1 may be the cells that developed into CD103+CD11b+ DC. To investigate this I analysed how CD101 and Trem-1 expression changed with age amongst the DC subsets in SI LP, colonic LP (CLP) and MLN. The proportion of CD101+Trem-1+ cells increased amongst CD103+CD11b+ DC in the SI LP and MLN with age, while amongst CD103+CD11b+ DC in the CLP this decreased. This was not the same in CD103-CD11b+ DC, where CD101 and Trem-1 expression was more varied with age in all tissues. CD101 and Trem-1 were not expressed to any great extent on CD103+CD11b- or CD103-CD11b- DC. The phenotypic development of the 19 intestinal DC subsets was paralleled by the gradual upregulation of CD103 expression, while the production of retinoic acid (RA), as assessed by the AldefluorTM assay, was low early in life and did not attain adult levels until after weaning. Thus DC in the neonatal intestine take some time to acquire the adult pattern of phenotypic subsets and are functionally immature compared with their adult counterparts. In Chapter 5, I used CD101 and Trem-1 to explore the ontogeny of intestinal DC subsets in CCR2-/- and SIRPα mt mice, both of which have selective defects in one particular group of DC. The selective defect seen amongst CD103+CD11b+ DC in adult SIRPα mt mice was more profound in mice at D7 and D14 of age, indicating that it may be intrinsic to this population and not highly dependent on environmental factors that change after birth. The expression of CD101 and Trem-1 by both CD103+CD11b+ and CD103-CD11b+ DC was reduced in SIRPα mt mice, again indicating that this entire lineage was affected by the lack of SIRPα signalling. However there was also a generalised defect in the numbers of all DC subsets in many tissues from early in life, suggesting there was compromised development, recruitment or survival of DC in the absence of SIRPα signalling. In contrast to the findings in SIRPα mt mice, more CD103+CD11b+ DC co-expressed CD101 and Trem-1 in CCR2-/- mice, while there were no differences in the expression of these molecules amongst CD103-CD11b+ DC. This may suggest that CCR2+ CD103-CD11b+ DC are not the cells that express CD101 and Trem-1 that are predicted to be the direct precursors of CD103+CD11b+ DC. I also examined the expression of DC growth factor receptors on DC subsets from mice of different ages, but no clear age or subset- related patterns of the expression of mRNA for Csf2ra, Irf4, Tgfbr1 and Rara could be observed. Next, I investigated whether Trem-1 played any role in DC development. Preliminary experiments in Trem-1-/- mice show no differences between any of the DC subsets, nor were there any selective effects on individual subsets when DC development from Trem-1-/- KO and WT BM was compared in competitive chimeras. However these experiments were difficult to interpret due to viability problems and because I found an unexpected defect in the ability of Trem-1-/- BM to generate all DC, irrespective of whether they expressed Trem-1 or not. 20 The final experiments I carried out were to examine the role of the microbiota in driving the differentiation of intestinal DC subsets, based on the hypothesis that this could be one of the environmental factors that might influence events in the developing intestine. To this end I performed experiments in both antibiotic treated and germ free adult mice, both of which showed no significant phenotypic differences amongst any of the DC subsets. However the study of germ free mice was compromised by recent contamination of the colony and may not be the conclusive answer. Together the data in this thesis have shown that the population of CD103+CD11b+ DC, which is unique to the intestine, is not present at birth. These cells gradually increase in frequency over time and as this occurs there is a reciprocal decrease in the frequency of CD103-CD11b+ DC. Along with other results, this leads to the idea that there may be a linear developmental pathway from CD103-CD11b+ DC to CD103+CD11b+ DC that is driven by non-microbial factors that are located preferentially in the small intestine. My project indicates that markers such as CD101 and Trem-1 may assist the dissection of this process and highlights the importance of the neonatal period for these events.

Assessments of training load in elite youth soccer

One of the most popular sports globally, soccer has seen a rise in the demands of the game over recent years. An increase in intensity and playing demands, coupled with growing social and economic pressures on soccer players means that optimal preparation is of paramount importance. Recent research has found the modern game, depending on positional role, to consist of approximately 60% more sprint distance in the English Premier League, which was also found to be the case for frequency and success of discrete technical actions (Bush et al., 2015). As a result, the focus on soccer training and player preparedness is becoming more prevalent in scientific research. By designing the appropriate training load, and thus periodization strategies, the aim is to achieve peak fitness in the most efficient way, whilst minimising the risk of injury and illness. Traditionally, training intensity has been based on heart rate responses, however, the emergence of tracking microtechnology such as global positioning system (GPS) and inertial sensors are now able to further quantify biomechanical load as well as physiological stress. Detailed pictures of internal and external loading indices such as these then combine to produce a more holistic view of training load experience by the player during typical drills and phases of training in soccer. The premise of this research is to gain greater understanding of the physical demands of common training methodologies in elite soccer to support optimal match performance. The coaching process may then benefit from being able to prescribe the most effective training to support these. The first experimental chapter in this thesis began by quantify gross training loads of the pre-season and in-season phases in soccer. A broader picture of the training loads inherent in these distinct phases brought more detail as to the type and extent of external loading experienced by soccer players at these times, and how the inclusion of match play influences weekly training rhythms. Training volume (total distance) was found to be high at the start compared to the end of pre-season (37 kilometres and 28 kilometres), where high cardiovascular loads were attained as part of the conditioning focus. This progressed transiently, however, to involve higher-speed, acceleration and change-of-direction stimuli at the end of pre-season compared to the start and to that in-season (1.18 kilometres, 0.70 kilometres and 0.42 kilometres high-intensity running; with 37, 25 and 23 accelerations >3m/s2 respectively) . The decrease in volume and increase in maximal anaerobic activity was evident in the training focus as friendly matches were introduced before the competitive season. The influence of match-play as being a large physical dose in the training week may then determine the change in weekly periodisation and how resulting training loads applied and tapered, if necessary. The focus of research was then directed more specifically to the most common mode of training in soccer, that also featured regularly in the pre-season period in the present study, small-sided games (SSG). The subsequent studies examined numerous manipulations of this specific form of soccer conditioning, such as player numbers as well as absolute and relative playing space available. In contrast to some previous literature, changing the number of players did not seem to influence training responses significantly, although playing format in the possession style brought about larger effects for heart rate (89.9%HRmax) and average velocity (7.6km/h-1). However, the following studies (Chapters 5, 6 and 7) revealed a greater influence of relative playing space available to players in SSG. The larger area at their disposal brought about greater aerobic responses (~90%HRmax), by allowing higher average and peak velocities (>25km/h-1), as well as greater distance acceleration behaviour at greater thresholds (>2.8m/s2). Furthermore, the data points towards space as being a large determinant in strategy of the player in small-sided games (SSG), subsequently shaping their movement behaviour and resulting physical responses. For example, higher average velocities in a possession format (8km/h-1) reflects higher work rate and heart rate load but makes achieving significant neuromuscular accelerations at a high level difficult given higher starting velocities prior to the most intense accelerations (4.2km/h-1). By altering space available and even through intentional numerical imbalances in team numbers, it may be easier for coaches to achieve the desired stimulus for the session or individual player, whether that is for aerobic and neuromuscular conditioning. Large effects were found for heart rate being higher in the underloaded team (85-90%HRmax) compared to the team with more players (80-85%HRmax) as well as for RPE (5AU versus 7AU). This was also apparent for meterage and therefore average velocity. It would also seem neuromuscular load through high acceleration and deceleration efforts were more pronounced with less numbers (given the need to press and close down opponents, and in a larger area relative to the number of players on the underloaded team. The peak accelerations and deceleration achieved was also higher when playing with less players (3-6.2m/s2 and 3-6.1m/s2) Having detailed ways in which to reach desired physical loading responses in common small training formats, Chapter 8 compared SSG to larger 9v9 formats with full-size 11v11 friendly matches. This enabled absolute and relative comparisons to be made and to understand the extent to which smaller training formats are able to replicate the required movements to be successful in competition. In relative terms, it was revealed that relative acceleration distance and Player Load were higher in smaller 4v4 games than match-play (1.1m.min-1 and 0.3m.min-1 >3m/s2; 16.9AU versus 12AU). Although the smallest format did not replicate the high-velocity demands of matches, the results confirmed their efficacy in providing significant neuromuscular load during the training week, which may then be supplemented by high-intensity interval running in order to gain exposure to more maximal speed work. In summary, the data presented provide valuable information from GPS and inertial sensor microtechnology which may then be used to understand training better to manipulate types of load according to physical conditioning objectives. For example, a library of resources to direct planning of drills of varying cardiovascular, neuromuscular and perceptual load can be created to give more confidence in session outcomes. Combining external and internal load data of common soccer training drills, and their application across different phases and training objectives may give coaches a powerful tool to plan and periodize training.