Investigating the impact of the parasite derived peptide FhHDM-1 on β-cell survival and function

University of Technology Sydney. Faculty of Science.Type 1 diabetes (T1D) is an autoimmune disease characterized by the irreversible immune-mediated destruction of the insulin producing β-cells within the pancreatic islets. Fasciola hepatica helminth defence molecule-1 (FhHDM-1), was previously identified as a parasite-derived peptide that permanently prevented T1D development in non-obese diabetic (NOD) mice. Disease prevention was associated with increased insulin content and decreased insulitis within the islet, however the exact protective mechanisms have yet to be elucidated. Therefore, the aim of this project was to determine whether this beneficial effect of FhHDM-1 was mediated by alterations to the survival and/or function of pancreatic β-cells. Initial analysis revealed that FhHDM-1 localised to the pancreas in vivo and directly interacted with β-cells in vitro to promote survival/function and prevent apoptosis in the presence of pro-inflammatory cytokines, without inducing proliferation. These positive effects were associated with the activation of PI3K/Akt signaling. Investigation of the underlying transcriptome and proteome changes induced by FhHDM-1 that led to activation of PI3K/Akt signaling identified insulin growth factor 1 receptor (IGF1R) as the probable initiating factor. However, despite an increase in expression and protein abundance of this receptor in FhHDM-1 treated β-cells, no direct interaction between FhHDM-1 and IGF1R occurred. Instead, FhHDM-1 treatment enhanced the secretion of IGF-2, in turn increasing the total abundance and phosphorylation of IGF1R at the tyrosine residue 1316. This phosphorylation site is associated with the recruitment of PI3K to IGF1R, and subsequent activation of the PI3K/Akt signaling cascade, in turn leading to enhanced β-cell survival and function. Since IGF-2 is not stored in the intracellular vesicles of β-cells, the data indicated that FhHDM-1 was activating the transcription of IGF-2 to mediate its positive downstream effects. This was confirmed by analysis of the miRNA-gene regulation within FhHDM-1 treated β-cells, which identified several miRNAs that correlated with IGF-2/PI3K/Akt modulation (such as upregulation of miR-483-3p and miR-124-3p, and downregulation of miR-466i-5p, miR-7689-3p, miR-30d-5p, miR-3470a, miR-677-5p, and miR-7033-5p). The collective outcomes of this research demonstrate that FhHDM-1 would be efficacious across multiple pathogeneses in which preservation of functional β-cell mass is required such as in individuals who are at-risk of T1D, in individuals that have undergone seroconversion, in recently diagnosed T1D patients with residual β-cell mass, in patients undergoing islet transplantation, and putatively in T2D, in which inflammation compromises β-cell function. Accordingly, FhHDM-1 represents a potential cure for a compilation of diseases that are the fastest growing and most chronic conditions worldwide

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Evaluation of protective immune responses against <em>Litomosoides sigmodontis</em> and analysis of <em>L. sigmodontis</em> extract on T cell modulation and glucose tolerance in diet-induced obese mice

Parasitic helminths are responsible for numerous tropical diseases which represent a major health problem in tropical and subtropical regions such as lymphatic filariasis (elephantiasis), causing lymphedema in limbs and scrotum in lymphatic filariasis patients and onchocerciasis (river blindness) leading to blindness and severe dermatitis in onchocerciasis patients. Helminths modulate the immune system of their hosts to their benefit, enabling and prolonging parasite survival in the host. On the other hand, several human and animal studies confirmed a protective effect of helminth infections and worm extracts on allergic and autoimmune diseases and more recently on metabolic diseases such as diet-induced insulin resistance. In order to obtain a better understanding of filarial immunomodulation and protective immune responses against filariae, the Litomosoides sigmodontis model was used. As type 2 immune responses are in general associated with protective immune responses against filariae, the present thesis directly assessed the role of eosinophils, IL-5 and IL-4R on L. sigmodontis infection. Therefore, wild-type (WT) BALB/c mice and mice deficient for eosinophils (dblGATA), IL-5, IL-4R and both IL-5/IL-4R were used. Analyses were performed at the peak of microfilaremia (71 days post infection (dpi)) and during a late time point of infection (119dpi). Following necropsy parasitological (worm and microfilaria load) as well as immunological parameters (cytokine levels, major immune cell types) within the thoracic cavity (the site of adult worm residence) and the spleen were analysed. A negative correlation between thoracic cavity eosinophil numbers and the adult worm burden as well as microfilaremia was observed. Alternatively activated macrophages (AAMs) correlated negatively with microfilaremia and positively with the adult worm load, which was associated with an extended adult worm survival in dblGATA, IL-5-/- and IL-4R-/-/IL-5-/- mice. Mice deficient for IL-4R/IL-5 had the highest susceptibility for L. sigmodontis infection, an earlier onset of microfilaremia, highest microfilariae (MF) loads and an extended adult worm survival. Another focus of this dissertation was the role of the damage-associated proteins S100A8/A9 during L. sigmodontis infection. S100A8/A9 was shown to provide an anti inflammatory effect within the lung during the migratory phase of infective L3 larvae. Therefore, S100A8/A9-/- mice exhibited a decreased worm burden, which was associated with an increased inflammatory immune response within the lung compared to WT controls. The reduced worm burden in S100A8/A9-/- mice was also given following subcutaneous (s.c.) infection and the immune response within the skin was not altered in comparison to WT controls. The S100A8/A9-mediated protective mechanisms occurred in the lung via neutrophils, but not in the skin. Furthermore, the impact of the aryl hydrocarbon receptor, belonging to the family of Pattern Recognition Receptors (PRRs), during L. sigmodontis infection was analyzed. AhR-/- mice exhibited an increased worm burden, which was presumably based on limited protective immune responses within the skin and increased vascular permeability, facilitating larval migration and parasite survival. The next part of this thesis studied the beneficial immunomodulatory effect of L. sigmodontis adult worm extract (LsAg) during diet-induced glucose intolerance and T cell inflammation. Repeated intraperitoneal (i.p.) injections of LsAg improved glucose tolerance, but not insulin sensitivity in obese mice. The protective effect was associated with a decreased type 1 inflammation in adipocytes, which represent a main cause of insulin resistance. IFN-γ, TNF and IL-17 were decreased in CD4+ T cells in LsAg treated mice. Furthermore TNF and IL-17 were significantly lower in CD8+ T cells in LsAg-treated animals compared to corresponding controls. Our study revealed that filarial extracts present potential candidates to treat diet induced insulin resistance. Finally, I contributed to the identification of novel macrofilaricidal compounds by in vivo testing in the L. sigmodontis rodent model with several industry partners.Parasitäre Helminthen sind für eine Vielzahl von tropischen Krankheiten verantwortlich, die ein großes Gesundheitsproblem in den Tropen und Subtropen darstellen. Bei der lymphatischen Filariose (Elephantiasis) leiden Patienten unter Lymphödemen in den Gliedmaßen und bei der Onchozerkose (Flussblindheit) unter schwerwiegender Dermatitis sowie Blindheit. Helminthen modulieren das Immunsystem ihres Wirtes zu ihrem Vorteil, welches das langfristige Überleben des Parasiten im Wirt ermöglicht. Zudem belegen humane und tierexperimentelle Studien, dass Helmintheninfektionen und -extrakte Allergien, Autoimmunerkrankungen sowie metabolische Krankheiten lindern und sogar verhindern können. Neue Therapieansätze gegen Filarieninfektionen, Allergien und Autoimmunerkrankungen werden mittels des Models L. sigmodontis untersucht und identifiziert. In der vorliegenden Arbeit wurde der direkte Einfluss von Eosinophilen, IL-5 und IL-4R auf die L. sigmodontis Infektion in WT BALB/c Mäusen und Mauslinien, welche defizient für Eosinophile (dblGATA), IL-5, IL-4R und IL-5/IL-4R sind, untersucht. Parasitologische sowie immunologische Parameter wurden während des Mikrofilarienpeaks (71dpi) und zu einem späten Zeitpunkt der Infektion (119dpi) analysiert. In der Pleuralhöhle befindliche Eosinophile korrelierten negativ mit der adulten Wurm- und Mikrofilarienlast. Alternativ aktivierte Makrophagen korrelierten ebenfalls negativ mit der Mikrofilarämie, jedoch positiv mit der adulten Wurmlast, die mit einer erhöhten Lebenserwartung der adulten Würmer in den Eosinophil-defizienten Mauslinien assoziiert war. Dabei konnte festgestellt werden, dass die IL-4R/IL5 defizienten Tiere alle MF positiv waren. Zusätzlich wiesen diese Tiere die höchste Mikrofilarienlast auf, die zusätzlich mit einer frühzeitigen Mikrofilarienfreisetzung assoziert war. Des Weiteren wurde im Rahmen dieser Dissertation eine anti-inflammatorische Rolle für S100A8/A9 in der Lunge während der L. sigmodontis Infektion nachgewiesen S100A8/A9-/- Mäuse waren durch eine geringere Adultwurmlast charakterisiert, die zusätzlich mit einer verstärkten inflammatorischen Immunantwort in der Lunge im Vergleich zu den WT Kontrollen assoziiert war. Subkutane und intradermale Injektionen von infektiösen L3 Larven in S100A8/A9-/- Mäusen hatten keinen Einfluss auf die Infektionslast. Dies zeigt, dass S100A8/A9-assozierte Immunantworten in der Haut nicht die Wurmlast beeinflussen. Zudem konnte gezeigt werden, dass ein durch Neutrophile initiierter protektiver Mechanismus in der Lunge stattfindet, da die Depletion von Neutrophilen in der Lunge in einer erhöhten Wurmlast in S100A8/A9-/- Tieren resultierte. Ein weiterer Teil der Dissertation beschäftigte sich mit dem Einfluss des Aryl Hydrocarbon Rezeptors (AhR) auf die L. sigmodontis Infektion. AhR-/- Mäuse waren durch eine erhöhte Wurmlast charakterisiert, die vermutlich auf eingeschränkte Immunantworten innerhalb der Haut, der Pleuralhöhle sowie einer erhöhten vaskulären Permeabilität zurückzuführen ist. Zusätzlich wurde in dieser Thesis der Effekt von adultem L. sigmodontis Wurmextrakt während einer Diät-induzierten Glukoseintoleranz auf die Zytokinproduktion von T Zellen untersucht. Intraperitoneale Injektionen von LsAg verbesserten die Glukosetoleranz, jedoch hatten keinen Einfluss auf die Insulintoleranz in adipösen Mäusen. Dieser protektive Effekt war mit einer verminderten Typ 1 Inflammation in den Adipozyten assoziiert. Darüber hinaus resultierte die Behandlung mit LsAg in einer verringerten pro-inflammatorischen Zytokinproduktion in CD4+ (IFNγ, TNF, IL-17) und CD8+ T Zellen (TNF, IL-17) im Vergleich zu PBS behandelten adipösen Kontrolltieren. Zusammengefasst stellen Filarienextrakte vielversprechende neue Therapieansätze für die Diät-induzierte Insulinresistenz dar. Letztendlich unterstütze ich die Identifizierung neuer makrofilarizider Substanzen im L. sigmodontis Nagetiermodell, die in enger Zusammenarbeit mit diversen Industriepartnern durchgeführt wurden

A ROLE FOR BACTERIAL-DERIVED PROTEASES AND PROTEASE INHIBITORS IN FOOD SENSITIVITY.

Celiac disease is a chronic atrophic enteropathy triggered by the ingestion of dietary gluten in genetically predisposed individuals expressing HLA class II genes, DQ2 or DQ8. Both innate and adaptive immune mechanisms are required for the development of the disease. The adaptive immune response is well characterized and includes the development of gluten-specific T-cells and antibodies towards gluten and the autoantigen, tissue transglutaminase 2. Less is known about the initiation of the innate immune response and its triggers, which is characterized by increases in cytokines such as IL-15, leading to proliferation and cytotoxic transformation of intraepithelial lymphocytes that are not specific to gluten. Although genetic susceptibility is necessary for celiac disease and present in 30% of most populations, only about 1% will develop it. This points to additional environmental factors, which could be microbial in origin. Disruptions of the gut microbial ecosystem, termed the intestinal microbiota, have been associated with the development and severity of many gastrointestinal disorders. Indeed, compositional differences in fecal and small intestinal microbiota have been described in patients with active celiac disease compared to healthy controls. This “dysbiosis” includes increases in small intestinal Proteobacteria and reductions in Firmicutes, but mechanistic information is lacking. Our lab has previously demonstrated that patients with active celiac disease have decreased expression of the serine protease inhibitor elafin in the duodenal mucosa, suggesting that proteolytic imbalance from either host or microbial origin, could be important in disease pathogenesis. The aim of this thesis is to study mechanisms through which the intestinal microbiota may contribute to the development of gluten sensitivity. I thus hypothesized that the proteolytic capacity of a dysbiotic small intestinal microbiota could promote the development of gluten sensitivity, in a genetically susceptible host. Firstly, I investigated whether the intestinal microbiota is a source of proteases in the gut lumen, that metabolize gluten in vivo affecting its immunogenicity. Secondly, I studied whether and how bacterial proteases stimulate innate immune mechanisms, relevant to celiac disease pathogenesis. Last, I investigated whether some of these mechanisms could be targeted to improve gluten sensitivity. In Chapter 3 of this thesis, I show that the small intestinal microbiota participates in gluten metabolism in vivo in mice. Proteobacteria and opportunistic pathogen Pseudomonas aeruginosa, isolated from patients with celiac disease, metabolize gluten proteins through microbial elastase. Bacterially-modified gluten peptides translocate the mucosal barrier with efficiency and retain immunostimulatory capacity when incubated with gluten-specific T-cells from patients with celiac disease. However, bacteria found in higher abundance in healthy individuals, such as Lactobacilli, metabolize P. aeruginosa modified gluten peptides reducing their antigenicity. This constitutes an opportunistic pathogen-gluten-host mechanism that could modulate celiac disease risk in genetically susceptible people. In Chapter 4, I show that bacterial proteases capable of extracellularly metabolizing gluten, such as P. aeruginosa elastase, also have the capacity to stimulate innate immune responses in the small intestine, likely through a protease activated receptor-2-dependent mechanism. This innate immune response does not require HLArisk genes and is characterized by non-specific increases in small intestinal intraepithelial lymphocytes. In mice transgenic for the HLA-DQ8 gene, the presence of P. aeruginosa elastase in the small intestine exacerbates gluten-induced pathology. In Chapter 5, I use a commensal Bifidobacterium longum strain that naturally produces a serine protease inhibitor, with the ability to inhibit elastase activity. I show that administration of Bifidobacterium longum to mice expressing the HLA-DQ8 gene prevents gluten immunopathology. The effect is dependent on the production of the bacterial serine protease inhibitor, as it was not observed with a B. longum knock-out for the serine protease inhibitor gene. Within this thesis, I demonstrate that microbial proteases can modulate gluten sensitivity through participation in both adaptive and innate immune pathways using celiac disease as a model of gastrointestinal disease. Further, I provide pre-clinical support that this pathway can be targeted for therapeutic purposes using protease inhibitors from commensal bacterial strains. My results provide causal evidence and novel mechanisms through which small intestinal bacteria may exacerbate or attenuate gluten sensitivity.ThesisDoctor of Philosophy (PhD

Viral infections in 47 CVID patients in allergy and immunology department of Rasool E Akram hospital in Tehran

Background: CVID is a heterogeneous primary immune deficiency with infectious, autoimmune and autoinflamatory features. It is most common symptomatic PID in Iran, with prevalence of 1 in 25000 to 50000 people. CVID has been divided into some phenotypes to produce more homogenized subpopulations. CVID is not a pure Ab deficiency .and because of both abnormalities in Tcell and innate immunity in combination with B cell dysfunction these patients are predisposed to viral and opportunistic infections. Method: prevalence of viral infections is reported in 47 CVID patients registered in Rasool E Akram hospital in Tehran. Patients have been diagnosed as CVID with the PAGID-ESID diagnostic criteria in our department or referred from other clinics for follow up and treatment. Diagnosis of viral germs has been made by clinical signs, pathological significances and in some cases by PCR. Cases: 9 patients (19%) had problems with viral infections. Infections occurred befor diagnosis of CVID in some cases or after that. Four patients (8.5 %) had problems with wart. Sever mucocutaneus HSV infection has occurred in 3 (6 %), recurrent zona in one (2 %) and CMV infection as colitis or pneumonitis in 3(6 %) patients. Sever progressive lethal CNS infection with JC virus occurred in one patient. Conclusion: evidences show that CVID is not a pure B cell defect, and we should be aware of opportunistic and viral infections that in some cases may be fatal