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.

The teaching of reading English in a foreign language in Libyan universities: methods and models

This study focuses on the learning and teaching of Reading in English as a Foreign Language (REFL), in Libya. The study draws on an action research process in which I sought to look critically at students and teachers of English as a Foreign Language (EFL) in Libya as they learned and taught REFL in four Libyan research sites. The Libyan EFL educational system is influenced by two main factors: the method of teaching the Holy-Quran and the long-time ban on teaching EFL by the former Libyan regime under Muammar Gaddafi. Both of these factors have affected the learning and teaching of REFL and I outline these contextual factors in the first chapter of the thesis. This investigation, and the exploration of the challenges that Libyan university students encounter in their REFL, is supported by attention to reading models. These models helped to provide an analytical framework and starting point for understanding the many processes involved in reading for meaning and in reading to satisfy teacher instructions. The theoretical framework I adopted was based, mainly and initially, on top-down, bottom-up, interactive and compensatory interactive models. I drew on these models with a view to understanding whether and how the processes of reading described in the models could be applied to the reading of EFL students and whether these models could help me to better understand what was going on in REFL. The diagnosis stage of the study provided initial data collected from four Libyan research sites with research tools including video-recorded classroom observations, semi-structured interviews with teachers before and after lesson observation, and think-aloud protocols (TAPs) with 24 students (six from each university) in which I examined their REFL reading behaviours and strategies. This stage indicated that the majority of students shared behaviours such as reading aloud, reading each word in the text, articulating the phonemes and syllables of words, or skipping words if they could not pronounce them. Overall this first stage indicated that alternative methods of teaching REFL were needed in order to encourage ‘reading for meaning’ that might be based on strategies related to eventual interactive reading models adapted for REFL. The second phase of this research project was an Intervention Phase involving two team-teaching sessions in one of the four stage one universities. In each session, I worked with the teacher of one group to introduce an alternative method of REFL. This method was based on teaching different reading strategies to encourage the students to work towards an eventual interactive way of reading for meaning. A focus group discussion and TAPs followed the lessons with six students in order to discuss the 'new' method. Next were two video-recorded classroom observations which were followed by an audio-recorded discussion with the teacher about these methods. Finally, I conducted a Skype interview with the class teacher at the end of the semester to discuss any changes he had made in his teaching or had observed in his students' reading with respect to reading behaviour strategies, and reactions and performance of the students as he continued to use the 'new' method. The results of the intervention stage indicate that the teacher, perhaps not surprisingly, can play an important role in adding to students’ knowledge and confidence and in improving their REFL strategies. For example, after the intervention stage, students began to think about the title, and to use their own background knowledge to comprehend the text. The students employed, also, linguistic strategies such as decoding and, above all, the students abandoned the behaviour of reading for pronunciation in favour of reading for meaning. Despite the apparent efficacy of the alternative method, there are, inevitably, limitations related to the small-scale nature of the study and the time I had available to conduct the research. There are challenges, too, related to the students’ first language, the idiosyncrasies of the English language, the teacher training and continuing professional development of teachers, and the continuing political instability of Libya. The students’ lack of vocabulary and their difficulties with grammatical functions such as phrasal and prepositional verbs, forms which do not exist in Arabic, mean that REFL will always be challenging. Given such constraints, the ‘new’ methods I trialled and propose for adoption can only go so far in addressing students’ difficulties in REFL. Overall, the study indicates that the Libyan educational system is underdeveloped and under resourced with respect to REFL. My data indicates that the teacher participants have received little to no professional developmental that could help them improve their teaching in REFL and skills in teaching EFL. These circumstances, along with the perennial problem of large but varying class sizes; student, teacher and assessment expectations; and limited and often poor quality resources, affect the way EFL students learn to read in English. Against this background, the thesis concludes by offering tentative conclusions; reflections on the study, including a discussion of its limitations, and possible recommendations designed to improve REFL learning and teaching in Libyan universities.