Infections are a common co-morbidity in Alzheimer's disease (AD), and evidence suggests that infections can exacerbate neuroinflammation and increase cognitive decline in AD patients. In AD, immune changes are observed both in the central nervous system (CNS) and in the rest of the body. However, only a few studies have investigated immune responses to infection in AD. Here, two extensively studied infections, Toxoplasma gondii (T. gondii) and Trichuris muris (T. muris) were used to investigate infection in AD. T. gondii is a protozoan parasite which is common globally, including in the developed world where AD cases are increasing dramatically. Infection with T. gondii starts in the gut, before becoming systemic and then infecting the CNS, where the parasite forms a chronic cyst infection. In contrast, T. muris is a nematode parasite, which remains localised to the gut. Notably, T. gondii is known to alter neuroinflammation and behaviour. T. gondii forms cysts preferentially in the areas of the brain commonly affected by AD, such as the hippocampus, which therefore makes it an interesting model to study co-morbidity. AD is often associated with advanced age. As we age, our immune system declines, and an important unanswered question is whether age impacts on the immune response to infection. This is of particular significance when considering chronic infections such as T. gondii, which require immune surveillance to prevent parasite recrudescence. Therefore, the aim of this thesis was to investigate infection in AD by determining: whether the immune response to an infection is altered in AD; whether the immune response to an infection in AD differs with age; what the effects of infection are on neuroinflammation, pathology and behaviour in AD; what are the effects of chronic infection with T. gondii. Immune responses to infection were altered in both the 3xTg-AD and the APP PS1 mouse models of AD, including increased inflammation and weight loss in AD mice following infection. Although older (eleven to twelve-month-old) 3xTg-AD mice showed some alterations in cytokine responses following infection, overall there were no major difference compared to younger (five to six-month-old) animals. Additionally, infection was found to alter neuroinflammation in both 3xTg-AD and APP PS1 mice, though differently. In 3xTg-AD mice, microglia activation increased following infection with T. gondii and T. muris, showing that infection did not need to be in the brain to alter neuroinflammation. In APP PS1 mice, a decrease in microglia activation occurred after infection with T. gondii, which was accompanied by an increase in IL-1alpha production and increased amyloid beta levels in APP PS1 mice following infection. However, no changes were found in behaviour following infection with T. gondii or T. muris in AD mouse models. Finally, chronic T. gondii infection was investigated in the TgF344-AD rat, which was established as a suitable AD model with both amyloid and tau pathology in which to study chronic infection. This work adds to a growing body of literature to suggest that infections are detrimental to AD patients, and that future measures to decrease morbidity could focus on further study of infections in AD, and the development of strategies to better prevent infections in AD patients.

The roles of CD4+ and CD8+ T-cells were studied by using Cryptosporidium muris and mouse model of infection: immunized and naive BALB/c mice as a source of cells for adoptive transfer of immunity to SCID mice. Naive and primed CD4+ T-cells have a major effect in immunity of cryptosporidial infection. However SCID mice reconstituted with primed CD8+ T-cells were able to recover from the infection. The purity of the adoptive transfer pre and post-inoculation is discussed.

Trichuris trichiura (T. Trichiura) is a large-intestinal dwelling nematode that affects over 1 billion people world-wide and thus has large global significance. Much of our understanding of T. trichiura infection comes from the study of the mouse model Trichuris muris (T. muris). However, how the immune system is initiated in response to helminth threat and how inflammation and pathology are resolved in T. muris infection still remain to be addressed. Here, I have attempted to provide insight into these questions. Previous work has shown resistance to T. muris infection is associated with the rapid recruitment of dendritic cells (DCs) to the colonic epithelium via epithelial production of CCL5 and CCL20. However, the epithelial-parasite interaction that drives chemokine production is not known. Pattern recognition receptor (PRRS) are critical mediators of pathogen recognition but there is no known (PRR) specific for T. muris. Here, we address the role of the cytosolic pattern recognition receptor Nod2, the location of which within the crypts correlates with the T. muris niche. In WT mice, in response to infection, there was a rapid influx of CD103+CD11c+ DCs into the colonic epithelium, whereas, this recruitment was impaired in Nod2 /- animals. In vitro and in vivo experiments confirmed the impairment in DC recruitment in Nod2-/- mice was attributable to the epithelial compartment. Subsequent work revealed decreased production of epithelial chemokines in the absence of functional Nod2. Thus, we have shown a novel role for Nod2 in the initiation the immune response to T. muris. We next addressed how pathology is regulated during T. muris infection. Firstly we investigated the role of arginase and Arg1-expressing macrophages in regulating pathology. My data showed that, unlike other gastrointestinal helminths, arginase and Arg1-expressing macrophages are not essential for resistance to T. muris or effective resolution of helminth-induced inflammation. I also addressed the role of DCs in the resolution of infection. DCs can regulate immune responses via the anti-inflammatory cytokine IL-10 and induction of regulatory T cells (Treg). I used an IL 10flox/floxCD11cCre transgenic model in which mice have DCs that cannot make IL-10. I found no role for CD11c+ cell mediated IL-10 production in the regulation of pathogen induced pathology in chronic T. muris infection. In summary I have been able to identify factors in the initiation of immunity to T. muris namely epithelial expression of Nod2. However, as arginase, Arg1-expressing macrophages and DC derived IL-10 appeared to play a redundant role in T. muris infection, the question as to how infection induced inflammation is resolved remains elusive.

No description available.

Goblet cells in the mucosal layer of the gastrointestinal tract are the primary source of gel-forming mucins, representing front-line defense. Sterile alpha motif-pointed domain ETS family transcription factor (SPDEF) has a crucial role in terminal differentiation, proliferation and maturation of goblet cells. Gut microbiota is an integral part of our internal environment. In a murine model of intestinal helminthic infection Trichuris muris, the interaction between host microbiota and parasite was seen to play critical roles in immune defense. This interaction is mediated through various mechanisms, including Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptor signaling cascades. However, the precise role of intestinal microbiota and NOD/TLR signaling in regulating SPDEF is not yet understood. Hence, we investigated the role of SPDEF in intestinal goblet cell response, the role of helminth-microbiota axis and NOD/TLR signaling in modulating SPDEF during T. muris infection. Experiments were conducted in wild-type (SPDEF+/+) and SPDEF-deficient (SPDEF-/-) mice on BALB/c background at different timepoints of T. muris infection. We observed increased PAS+ goblet cells and higher expression of SPDEF and Muc2 in SPDEF+/+ mice following infection with elevated levels of IL-4 and IL-13. SPDEF+/+ mice showed decreased worm burden from day 14 to 21 post-infection. Microbial analysis revealed altered composition in SPDEF+/+ and SPDEF-/- after infection. Microbiota was transplanted from naïve and T. muris infected mice to separate groups of antibiotic-treated (ABX-treated) mice. Increased PAS+ goblet cells and higher expression of SPDEF and Muc2 were observed in ABX-treated mice after receiving naive and T. muris-altered microbiota. Goblet cell number, the expression of SPDEF and Muc2 were higher in ABX-treated mice who received T. muris-altered microbiota. Microbial analysis revealed differences in T. muris-altered microbiota compared to naïve microbiota. In vitro experiment was conducted in human colonic mucin secreting LS174T cells where we observed stimulated mRNA expression of SPDEF and MUC2 by T. muris excretory-secretory products. These findings reveal new information about major interactions among parasites, microbiota and SPDEF-mediated intestinal goblet cell response in the context of host defense. / Thesis / Master of Health Sciences (MSc)

No description available.

IL-1 is a pro-inflammatory cytokine which play an important role in the activation and regulation of host defence and immune responses to inflammation or injury. IL-1 is able to bind and activate IL1-RI and IL1-RII, which are found on many cells types. The role of the IL-1 signalling in the deployment of Th cell subsets, especially Th17 cells is well known. However, the specific cells which are responsible for the expression of IL-1 signalling in the immune defense and in the development of the Th cell lineage in response to infection, is still largely unclear. Therefore in this thesis, IL1-RI conditional knockout mice specifically in hematopoietic cells (IL1-RI vaviCre+) were generated. Using IL1-RI vaviCre+ mice in comparison with IL1-RI global knockout mice (IL1-RI-/-) would determine whether the expression IL-1 signalling from hematopoietic cells is responsible for the immune defense and in the development of the Th1, Th2 and Th17 cells against gastrointestinal helminth Trichuris muris (T.muris) infections. The generation of IL1-RI vaviCre+ mice have been investigated at the genomic and proteomic level in order to confirm that the Il1-rI gene is inactivated in hematopoietic cells. The characterisation of IL1-RI vaviCre + mice at the genomic level confirmed that the Il1-rI gene was obliterated successfully. At protein level the characterisation of IL1- RI vaviCre + mice confirmed that IL1-RI was dysfunctional in hematopoietic cells. Additionally, the development of the immune cells was investigated in IL1-RI vaviCre + and IL1-RI-/- mice. Our findings demonstrated that the lymphocyte development was not affected by the deletion of the IL1- RI gene. This data indicated that IL1- RI vaviCre + and IL1-RI-/- mice are vital in vivo models. In high dose infection, both IL1-RI vaviCre + and IL1-RI -/- mice were able to clear the infections due to their ability to generate a Th2 response. Both IL1-RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris were susceptible to infections and showed high levels of Th1 cytokines. Thus, we hypothesised that IL1-RI signalling in hematopoietic cells was not required for worm expulsion and the generation of Th2 and Th1 response. Interestingly, low dose T.muris infection showed a clear reduction in the Th17 cytokines IL22 and IL17 in both IL1-RI vaviCre + and IL1-RI -/- mice, suggesting that IL-1 signalling expressed from hematopoietic cells is responsible for the development of Th17 cells and secretion of IL17 and IL22. IL1- RI vaviCre + and IL1-RI -/- mice infected with low dose of T.muris also showed an increase in inflammation in the colon and decreased of goblet cell hyperplasia. It is well known that IL22 plays an important role in preventing tissue damage and repair. Thus, in this study IL22 global knockout mice (IL22 -/-) were used to determine if the change in crypt lengths and goblet cell hyperplasia in IL1-RI vaviCre + and IL1-RI -/- was due to an absence of IL22. Our finding showed that IL22 -/- mice infected with low dose of T.muris had increased crypt length and a reduction in goblet cells. The similar phenotype in crypt length and goblet cell hyperplasia between IL22 -/-, IL1-RI vaviCre + and IL1-RI -/- mice suggested that a lack of IL22 in IL1-RI vaviCre + and IL1-RI -/- mice is responsible for the change in mice phenotype. It also provides more evidence for the role of IL-1 signaling in hematopoietic cells in the generation of Th17 cells and in the production of its cytokine IL22.IL1-RII is an inhibitor of IL1-RI, thus, in this study IL1-RII global knockout mice (IL1-RII -/-) mice was used in comparison with IL1-RI -/- mice to verify the role of IL-1 signaling in the development of Th17 cells. Our finding showed an overexpression of IL17 and IL22 in IL1-RII -/- compared with IL1-RI -/- mice and a higher level of IL17 in IL1-RII -/- mice compared with IL1-RII flox/flox mice. This data confirmed that IL-1 signaling is important for the development of Th17 cells and the production of its cytokine IL17 and IL22.

The data presented within this thesis shows the generation and characterisation of a complete-, macrophage/granulocyte- and T cell-specific IFNγR2 deficient mouse mutant. This mutant mouse is a valuable tool in dissecting the mechanism of action of the pleiotrophic cytokine IFNγ.The global mutant mouse was tested in three models in vivo - DSS induced colitis, Trichuris muris infection and EAE. The aim of the DSS-induced colitis model was to test the role of IFNγ in the innate immune system and, despite previous reports demonstrating IFNγ deficient mice are protected from DSS-colitis, our IFNγR2 deficient mice displayed equal or more severe colitis than control mice. We hypothesise that this discrepancy is due to differences in the gut microbiota.The Trichuris muris model was utilised as a method of examining the role of IFNγ in the adaptive immune system. The complete IFNγR2 mutant was resistant to a low dose T. muris infection; however, neither the T cell specific nor the macrophage/granulocyte specific mutant duplicated the resistant phenotype observed in the global knock-out mice. Analysis of a double conditional T cell and macrophage/granulocyte specific IFNγR2 mutant produced inconsistent results. Initial experiments suggested that, in combination, these deficiencies are sufficient to duplicate the resistant phenotype observed in the global mutant mice, but this was not reproducible.The final in vivo model that we used to analyse IFNγR2 mutant mice was EAE. This model was chosen as, for a long time, the mechanism of action and the involvement of IFNγ in EAE has been a matter of uncertainty. These results demonstrated that global IFNγR2 mutant mice demonstrate an atypical phenotype, with no signs of recovery. In contrast, control mice develop classical EAE symptoms with almost complete recovery prior to the termination of the experiment. The IFNγ receptor mutant mouse generated will be of great value to the scientific community as IFNγ has been demonstrated to play a role in multiple diseases and this tool allows the mechanism of action of this cytokine to be unravelled.

In the gastrointestinal (GI) tract, goblet cells are the major source of mucins, the main structural components of the mucus layer, which functions as the front line of innate defense. The GI tract contains trillions of commensal microbes, and these microbes can manipulate mucin production by activating different signalling cascades initiated by pattern recognition receptors (PRRs), including bacterial sensing Toll-like receptors (TLRs). In addition, sterile α motif pointed domain-containing ETS transcription factor (SPDEF) is a transcription factor that modulates goblet cell differentiation and positively regulates mucin production. During helminth infections, due to the co-existence of parasites and microbiota in close proximity of goblet cells in the gut, it is likely that helminth-microbiota interactions play an important role in mucin production. Indeed, goblet cell hyperplasia and increased mucin production are observed in many enteric helminth infections, including Trichuris muris, and these processes play key roles in host infection clearance. However, it should be noted that the role of microbiota within this axis is not yet understood. Here, we hypothesize T. muris-induced altered microbiota modulates goblet cell differentiation and mucin production via SPDEF-mediated transcriptional regulation and TLR2 signalling. C57BL/6 mice were gavaged with ~300 T. muris eggs. Mice were sacrificed 36 days post-infection. Microbiota from these T. muris-infected and non-infected mice were transferred into two groups of germ-free (GF) mice. Microbiota analysis revealed that treatments in both experiments (infection with T. muris and microbiota transfer from T. muris-infected mice into GF mice) significantly account for the among-sample variations in the composition of the gut microbiota between groups (p <= 0.001). In GF mice, transfer of T. muris-infected microbiota significantly increased goblet cell numbers and TLR2 expression as well as upregulated Muc2 expression compared to MSc Thesis –Yousefi Y; McMaster University – Medical Sciences v GF mice with non-infected microbiota. Antibiotic-treated (ABX-treated) TLR2 knockout (KO) mice after receiving microbiota from T. muris-infected mice showed significantly decreased expression of Muc2 and Muc5ac compared to ABX-treated wild-type (WT) mice receiving the same microbiota. To investigate whether SPDEF is a driving factor for Muc2 production in response to T. muris microbiota stimulation, we next transferred T. muris-infected microbiota into antibiotic-treated SPDEF KO and WT mice. We observed a slight, though not significant, the influence of SPDEF on the stimulation of mucin production by T. muris microbiota. These findings reveal important interactions among parasites, resident microbiota, and host in relation to goblet cell response in the gut. In addition, this study provides new information on TLR2-based innate signalling in the regulation of goblet cell biology and mucin productio / Thesis / Master of Science (MSc)

The development of immune response accountable for the ability to control Cryptosporidium muris TS03 infection was studied using immunocompetent and various types of immunodeficient mouse models. Subsequently the immune response was characterized by analysis of leukocyte infiltration and cytokine production in gastric epithelium. Moreover, the potentiality of immunocompetent mice to develop effective immune response to C. andersoni LI03 infection with consequent protection to consequent infection of the same mice with C. muris TS03 was also studied by monitoring oocysts shedding, leukocyte infiltration of the gastric mucosa and cytokine production in ex vivo cultures of splenocytes.