Increased tubulointerstitial recruitment of human CD141(hi) CLEC9A(+) and CD1c(+) myeloid dendritic cell subsets in renal fibrosis and chronic kidney disease

Dendritic cells (DCs) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated, and phenotyped them by multicolor flow cytometry. We detected significantly greater numbers of total DCs as well as CD141(hi) and CD1c(+) myeloid DC (mDCs) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy kidney tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared with healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141(hi) DCs expressed C-type lectin domain family 9 member A (CLEC9A), whereas the majority of CD1c(+) DCs lacked the expression of CD1a and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), suggesting these mDC subsets may be circulating CD141(hi) and CD1c(+) blood DCs infiltrating kidney tissue. Our analysis revealed CLEC9A(+) and CD1c(+) cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared with healthy tissue. Transforming growth factor-β levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared with nonfibrotic biopsies, with mDCs identified as a major source of this profibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and, thus, progression to chronic kidney disease.

Brief report: Intestinal dysbiosis in ankylosing spondylitis

Objective Ankylosing spondylitis (AS) is a common, highly heritable immune-mediated arthropathy that occurs in genetically susceptible individuals exposed to an unknown but likely ubiquitous environmental trigger. There is a close relationship between the gut and spondyloarthritis, as exemplified in patients with reactive arthritis, in whom a typically self-limiting arthropathy follows either a gastrointestinal or urogenital infection. Microbial involvement in AS has been suggested; however, no definitive link has been established. The aim of this study was to determine whether the gut in patients with AS carries a distinct microbial signature compared with that in the gut of healthy control subjects. Methods Microbial profiles for terminal ileum biopsy specimens obtained from patients with recent-onset tumor necrosis factor antagonist-naive AS and from healthy control subjects were generated using culture-independent 16S ribosomal RNA gene sequencing and analysis techniques. Results Our results showed that the terminal ileum microbial communities in patients with AS differ significantly (P < 0.001) from those in healthy control subjects, driven by a higher abundance of 5 families of bacteria (Lachnospiraceae [P = 0.001], Ruminococcaceae [P = 0.012], Rikenellaceae [P = 0.004], Porphyromonadaceae [P = 0.001], and Bacteroidaceae [P = 0.001]) and a decrease in the abundance of 2 families of bacteria (Veillonellaceae [P = 0.01] and Prevotellaceae [P = 0.004]). Conclusion We show evidence for a discrete microbial signature in the terminal ileum of patients with AS compared with healthy control subjects. The microbial composition was demonstrated to correlate with disease status, and greater differences were observed between disease groups than within disease groups. These results are consistent with the hypothesis that genes associated with AS act, at least in part, through effects on the gut microbiome.

The intestinal microbiome in human disease and how it relates to arthritis and spondyloarthritis

Humans and microbes have developed a symbiotic relationship over time, and alterations in this symbiotic relationship have been linked to several immune mediated diseases such as inflammatory bowel disease, type 1 diabetes and spondyloarthropathies. Improvements in sequencing technologies, coupled with a renaissance in 16S rRNA gene based community profiling, have enabled the characterization of microbiomes throughout the body including the gut. Improved characterization and understanding of the human gut microbiome means the gut flora is progressively being explored as a target for novel therapies including probiotics and faecal microbiota transplants. These innovative therapies are increasingly used for patients with debilitating conditions where conventional treatments have failed. This review discusses the current understanding of the interplay between host genetics and the gut microbiome in the pathogenesis of spondyloarthropathies, and how this may relate to potential therapies for these conditions.

The role of IL-17-secreting mast cells in inflammatory joint disease

The proinflammatory cytokine IL-17 has an important role in pathogenesis of several inflammatory diseases. In immune-mediated joint diseases, IL-17 can induce secretion of other proinflammatory cytokines such as IL-1, IL-6 and TNF, as well as matrix metalloproteinase enzymes, leading to inflammation, cartilage breakdown, osteoclastogenesis and bone erosion. In animal models of inflammatory arthritis, mice deficient in IL-17 are less susceptible to development of disease. The list of IL-17-secreting cells is rapidly growing, and mast cells have been suggested to be a dominant source of IL-17 in inflammatory joint disease. However, many other innate sources of IL-17 have been described in both inflammatory and autoinflammatory conditions, raising questions as to the role of mast cells in orchestrating joint inflammation. This article will critically assess the contribution of mast cells and other cell types to IL-17 production in the inflammatory milieu associated with inflammatory arthritis, understanding of which could facilitate targeted therapeutic approaches. © 2013 Macmillan Publishers Limited. All rights reserved.

Identification of 15 new psoriasis susceptibility loci highlights the role of innate immunity

To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of 3 genome-wide association studies (GWAS) and 2 independent data sets genotyped on the Immunochip, including 10,588 cases and 22,806 controls. We identified 15 new susceptibility loci, increasing to 36 the number associated with psoriasis in European individuals. We also identified, using conditional analyses, five independent signals within previously known loci. The newly identified loci shared with other autoimmune diseases include candidate genes with roles in regulating T-cell function (such as RUNX3, TAGAP and STAT3). Notably, they included candidate genes whose products are involved in innate host defense, including interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C) and nuclear factor (NF)-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense. © 2012 Nature America, Inc. All rights reserved.

Inflammation-driven bone formation in a mouse model of ankylosing spondylitis: Sequential not parallel processes

Background Ankylosing spondylitis (AS) is an immune-mediated arthritis particularly targeting the spine and pelvis and is characterised by inflammation, osteoproliferation and frequently ankylosis. Current treatments that predominately target inflammatory pathways have disappointing efficacy in slowing disease progression. Thus, a better understanding of the causal association and pathological progression from inflammation to bone formation, particularly whether inflammation directly initiates osteoproliferation, is required. Methods The proteoglycan-induced spondylitis (PGISp) mouse model of AS was used to histopathologically map the progressive axial disease events, assess molecular changes during disease progression and define disease progression using unbiased clustering of semi-quantitative histology. PGISp mice were followed over a 24-week time course. Spinal disease was assessed using a novel semi-quantitative histological scoring system that independently evaluated the breadth of pathological features associated with PGISp axial disease, including inflammation, joint destruction and excessive tissue formation (osteoproliferation). Matrix components were identified using immunohistochemistry. Results Disease initiated with inflammation at the periphery of the intervertebral disc (IVD) adjacent to the longitudinal ligament, reminiscent of enthesitis, and was associated with upregulated tumor necrosis factor and metalloproteinases. After a lag phase, established inflammation was temporospatially associated with destruction of IVDs, cartilage and bone. At later time points, advanced disease was characterised by substantially reduced inflammation, excessive tissue formation and ectopic chondrocyte expansion. These distinct features differentiated affected mice into early, intermediate and advanced disease stages. Excessive tissue formation was observed in vertebral joints only if the IVD was destroyed as a consequence of the early inflammation. Ectopic excessive tissue was predominantly chondroidal with chondrocyte-like cells embedded within collagen type II- and X-rich matrix. This corresponded with upregulation of mRNA for cartilage markers Col2a1, sox9 and Comp. Osteophytes, though infrequent, were more prevalent in later disease. Conclusions The inflammation-driven IVD destruction was shown to be a prerequisite for axial disease progression to osteoproliferation in the PGISp mouse. Osteoproliferation led to vertebral body deformity and fusion but was never seen concurrent with persistent inflammation, suggesting a sequential process. The findings support that early intervention with anti-inflammatory therapies will be needed to limit destructive processes and consequently prevent progression of AS.

Genetics of ankylosing spondylitis - Insights into pathogenesis

Ankylosing spondylitis (AS), an immune-mediated arthritis, is the prototypic member of a group of conditions known as spondyloarthropathies that also includes reactive arthritis, psoriatic arthritis and enteropathic arthritis. Patients with these conditions share a clinical predisposition for spinal and pelvic joint dysfunction, as well as genetic associations, notably with HLA-B*27. Spondyloarthropathies are characterized by histopathological inflammation in entheses (regions of high mechanical stress where tendons and ligaments insert into bone) and in the subchondral bone marrow, and by abnormal osteoproliferation at involved sites. The association of AS with HLA-B*27, first described >40 years ago, led to hope that the cause of the disease would be rapidly established. However, even though many theories have been advanced to explain how HLA-B*27 is involved in AS, no consensus about the answers to this question has been reached, and no successful treatments have yet been developed that target HLA-B27 or its functional pathways. Over the past decade, rapid progress has been made in discovering further genetic associations with AS that have shed new light on the aetiopathogenesis of the disease. Some of these discoveries have driven translational ideas, such as the repurposing of therapeutics targeting the cytokines IL-12 and IL-23 and other factors downstream of this pathway. AS provides an excellent example of how hypothesis-free research can lead to major advances in understanding pathogenesis and to the development of innovative therapeutic strategies.

Genetic analysis of chromosomal regions 2q33, 7q32 and 19q13 in multiple sclerosis susceptibility

Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS) affecting 0.1-0.2% of Northern European descent population. MS is considered to be a multifactorial disease, both environment and genetics play a role in its pathogenesis. Despite several decades of intense research, the etiological and pathogenic mechanisms underlying MS remain still largely unknown and no curative treatment exists. The genetic architecture underlying MS is complex with multiple genes involved. The strongest and the best characterized predisposing genetic factors for MS are located, as in other immune-mediated diseases, in the major histocompatibility complex (MHC) on chromosome 6. In humans MHC is called human leukocyte antigen (HLA). Alleles of the HLA locus have been found to associate strongly with MS and remained for many years the only consistently replicable genetic associations. However, recently other genes located outside the MHC region have been proposed as strong candidates for susceptibility to MS in several studies. In this thesis a new genetic locus located on chromosome 7q32, interferon regulatory factor 5 (IRF5), was identified in the susceptibility to MS. In particular, we found that common variation of the gene was associated with the disease in three different populations, Spanish, Swedish and Finnish. We also suggested a possible functional role for one of the risk alleles with impact on the expression of the IRF5 locus. Previous studies have pointed out a possible role played by chromosome 2q33 in the susceptibility to MS and other autoimmune disorders. The work described here also investigated the involvement of this chromosomal region in MS predisposition. After the detection of genetic association with 2q33 (article-1), we extended our analysis through fine-scale single nucleotide polymorphism (SNP) mapping to define further the contribution of this genomic area to disease pathogenesis (article-4). We found a trend (p=0.04) for association to MS with an intronic SNP located in the inducible T-cell co-stimulator (ICOS) gene, an important player in the co-stimulatory pathway of the immune system. Expression analysis of ICOS revealed a novel, previously uncharacterized, alternatively spliced isoform, lacking the extracellular domain that is needed for ligand binding. The stability of the newly-identified transcript variant and its subcellular localization were analyzed. These studies indicated that the novel isoform is stable and shows different subcellular localization as compared to full-length ICOS. The novel isoform might have a regulatory function, but further studies are required to elucidate its function. Chromosome 19q13 has been previously suggested as one of the genomic areas involved in MS predisposition. In several populations, suggestive linkage signals between MS predisposition and 19q13 have been obtained. Here, we analysed the role of allelic variation in 19q13 by family based association analysis in 782 MS families collected from Finland. In this dataset, we were not able to detect any statistically significant associations, although several previously suggested markers were included to the analysis. Replication of the previous findings on the basis of linkage disequilibrium between marker allele and disease/risk allele appears notoriously difficult because of limitations such as allelic heterogeneity. Re-sequencing based approaches may be required for elucidating the role of chromosome 19q13 with MS. This thesis has resulted in the identification of a new MS susceptibility locus (IRF5) previously associated with other inflammatory or autoimmune disorders, such as SLE. IRF5 is one of the mediators of interferons biological function. In addition to providing new insight in the possible pathogenetic pathway of the disease, this finding suggests that there might be common mechanisms between different immune-mediated disorders. Furthermore the work presented here has uncovered a novel isoform of ICOS, which may play a role in regulatory mechanisms of ICOS, an important mediator of lymphocyte activation. Further work is required to uncover its functions and possible involvement of the ICOS locus in MS susceptibility.

Identifying Genetic Risk Factors in Canine Autoimmune Disorders

Autoimmune diseases are more common in dogs than in humans and are already threatening the future of some highly predisposed dog breeds. Susceptibility to autoimmune diseases is controlled by environmental and genetic factors, especially the major histocompatibility complex (MHC) gene region. Dogs show a similar physiology, disease presentation and clinical response as humans, making them an excellent disease model for autoimmune diseases common to both species. The genetic background of canine autoimmune disorders is largely unknown, but recent annotation of the dog genome and subsequent development of new genomic tools offer a unique opportunity to map novel autoimmune genes in various breeds. Many autoimmune disorders show breed-specific enrichment, supporting a strong genetic background. Furthermore, the presence of hundreds of breeds as genetic isolates facilitates gene mapping in complex autoimmune disorders. Identification of novel predisposing genes establishes breeds as models and may reveal novel candidate genes for the corresponding human disorders. Genetic studies will eventually shed light on common biological functions and interactions between genes and the environment. This study aimed to identify genetic risk factors in various autoimmune disorders, including systemic lupus erythematosus (SLE)-related diseases, comprising immune-mediated rheumatic disease (IMRD) and steroid-responsive meningitis arteritis (SMRA) as well as Addison s disease (AD) in Nova Scotia Duck Tolling Retrievers (NSDTRs) and chronic superficial keratitis (CSK) in German Shepherd dogs (GSDs). We used two different approaches to identify genetic risk factors. Firstly, a candidate gene approach was applied to test the potential association of MHC class II, also known as a dog leukocyte antigen (DLA) in canine species. Secondly, a genome-wide association study (GWAS) was performed to identify novel risk loci for SLE-related disease and AD in NSDTRs. We identified DLA risk haplotypes for an IMRD subphenotype of SLE-related disease, AD and CSK, but not in SMRA, and show that the MHC class II gene region is a major genetic risk factor in canine autoimmune diseases. An elevated risk was found for IMRD in dogs that carried the DLA-DRB1*00601/DQA1*005011/DQB1*02001 haplotype (OR = 2.0, 99% CI = 1.03-3.95, p = 0.01) and for ANA-positive IMRD dogs (OR = 2.3, 99% CI = 1.07-5.04, p-value 0.007). We also found that DLA-DRB1*01502/DQA*00601/DQB1*02301 haplotype was significantly associated with AD in NSDTRs (OR = 2.1, CI = 1.0-4.4, P = 0.044) and the DLA-DRB1*01501/DQA1*00601/DQB1*00301 haplotype with the CSK in GSDs (OR=2.67, CI=1.17-6.44, p= 0.02). In addition, we found that homozygosity for the risk haplotype increases the risk for each disease phenotype and that an overall homozygosity for the DLA region predisposes to CSK and AD. Our results have enabled the development of genetic tests to improve breeding practices by avoiding the production of puppies homozygous for risk haplotypes. We also performed the first successful GWAS for a complex disease in dogs. With less than 100 cases and 100 controls, we identified five risk loci for SLE-related disease and AD and found strong candidate genes involved in a novel T-cell activation pathway. We show that an inbred dog population has fewer risk factors, but each of them has a stronger genetic risk. Ongoing studies aim to identify the causative mutations and bring new knowledge to help diagnostics, treatment and understanding of the aetiology of SLE-related diseases.

Impacto da exposição fetal ao HIV-1 na função das células T e das células dendríticas de neonatos não infectados

A síndrome da imunodeficiência adquirida (AIDS), causada pelo vírus da imunodeficiência humana (HIV), é uma das epidemias mais impactantes do milênio e, desde o início, o número de mulheres jovens infectadas vem aumentando vertiginosamente, principalmente nos países em desenvolvimento onde muitas destas engravidam precocemente. Apesar da grande maioria dos casos de AIDS pediátrico no mundo resultar da transmissão vertical, aproximadamente dois terços dos neonatos expostos ao HIV durante a vida fetal não são contaminados. Neste sentido, seguindo as recomendações do consenso brasileiro (Ministério da Saúde), toda criança cuja transmissão vertical tenha sido descartada laboratorialmente não necessita de acompanhamento ambulatorial particularizado. Entretanto, resultados anteriores obtidos pelo nosso grupo demonstraram que, gestantes infectadas pelo HIV-1 que não controlam a carga viral plasmática (CVP), apresentam níveis elevados de citocinas inflamatórias e, no presente estudo, resultados revelam que neonatos não-infectados nascidos dessas gestantes apresentam anormalidades imunofuncionais no compartimento das células T do cordão umbilical quando expostos in vitro a ativadores policlonais, mas não aos antígenos do HIV-1. Ademais, quando comparada a neonatos não expostos, a ativação in vitro das células T de neonatos expostos ao HIV-1 com anti-CD3/anti-CD28 induziu a produção de níveis elevados de IL-17 e reduzidos de IL-10. Interessantemente, essa tendência das células T em secretar IL-17 parece estar relacionada à liberação de níveis elevados de IL-23 pelas células dendríticas derivadas de monócitos do sangue do cordão umbilical estimuladas com lipopolissacarídeo bacteriano. Uma ausência de sensibilização uterina aos antígenos do HIV-1 sugere que essas alterações possam traduzir um efeito adverso da produção elevada de citocinas inflamatórias maternas sobre o sistema imune do neonato, o que pode desequilibrar os eventos envolvidos na maturação e homeostasia imune fetal e neonatal, favorecendo o predomínio de fenótipos Th anômalos, tal como Th17. Essa hiper-responsividade das células Th17 pode vir a comprometer não apenas a capacidade da criança em responder de forma adequada a diferentes estímulos antigênicos ao longo de sua vida, como também pode torná-la mais suscetível a desordens imunomediadas

Tryptophan degradation in irritable bowel syndrome: evidence of indoleamine 2,3-dioxygenase activation in a male cohort

Background: Irritable bowel syndrome (IBS) is a common disorder that affects 10–15% of the population. Although characterised by a lack of reliable biological markers, the disease state is increasingly viewed as a disorder of the brain-gut axis. In particular, accumulating evidence points to the involvement of both the central and peripheral serotonergic systems in disease symptomatology. Furthermore, altered tryptophan metabolism and indoleamine 2,3-dioxygenase (IDO) activity are hallmarks of many stress-related disorders. The kynurenine pathway of tryptophan degradation may serve to link these findings to the low level immune activation recently described in IBS. In this study, we investigated tryptophan degradation in a male IBS cohort (n = 10) and control subjects (n = 26). Methods: Plasma samples were obtained from patients and healthy controls. Tryptophan and its metabolites were measured by high performance liquid chromatography (HPLC) and neopterin, a sensitive marker of immune activation, was measured using a commercially available ELISA assay. Results: Both kynurenine levels and the kynurenine:tryptophan ratio were significantly increased in the IBS cohort compared with healthy controls. Neopterin was also increased in the IBS subjects and the concentration of the neuroprotective metabolite kynurenic acid was decreased, as was the kynurenic acid:kynurenine ratio. Conclusion: These findings suggest that the activity of IDO, the immunoresponsive enzyme which is responsible for the degradation of tryptophan along this pathway, is enhanced in IBS patients relative to controls. This study provides novel evidence for an immune-mediated degradation of tryptophan in a male IBS population and identifies the kynurenine pathway as a potential source of biomarkers in this debilitating condition.

Novel insights into the inflammatory basis of gastrointestinal disease

It has become clear that inflammation is beneficial to man, there are situations though that the inflammatory response causes damage to the host that is harmful to health. When the inflammatory response fails or is too strong, the health of the host is damaged and disease can occur. The implication of intestinal disease caused by an ineffective immune response is of great social and economic burden to society. The overarching purpose of this thesis is to assess inflammatory signalling targets associated with immune mediated disorders such as IBD, IBS and inflammatory liver disease. By assessing these targets and modifying their function I hope to contribute and expand further the pre-existing information on these disorders and improve the therapeutic interventions available in these debilitating conditions. I will assess the role of inflammation in disorders of the GI tract and liver IBD, IBS, hepatic inflammatory injury and furthermore, I will use pharmaceutical agents to activate and suppress components of the immune system. I will examine the inflammatory response in experimental models of disease for IBD and liver injury, I will attempt to alter these pathways using pharmaceutical intervention to delineate the disease causing mechanism that may lead to clinically relevant therapeutic interventions. In regards to IBS, I will attempt to improve the existing knowledge that exists in relation to the pathogenesis of this functional bowel disorder. I will attempt to define a mechanism by which the low grade mucosal inflammation that has been demonstrated by others arises and what this inflammation is induced by. The overall aim of this thesis is to attempt to further understand the mechanisms behind GI and liver disease. Looking at the inflammatory response in these specific conditions and how they can be altered may lead to exciting new therapies for inflammatory conditions in the gastrointestinal tract.

Molecular analysis of inflammatory mechanisms associated with Escherichia coli and inflammatory bowel diseases

Inflammatory bowel diseases (IBD), encompasses a range of chronic, immune-mediated inflammatory disorders that are usually classified under two major relapsing conditions, Crohn’s Disease (CD) and ulcerative colitis (UC). Extensive studies in the last decades have suggested that the etiology of IBD involves environmental and genetic factors that lead to dysfunction of epithelial barrier with consequent deregulation of the mucosal immune system and inadequate responses to gut microbiota.Over the last decade, the microbial species that has attracted the most attention, with respect to CD etiology, is Eschericia coli. In CD tissue, E. coli antigens have also been identified in macrophages within the lamina propria, granulomas, and in the germinal centres of mesenteric lymph nodes of patients. They have been shown to adhere to and invade intestinal epithelial cells whilst also being able to extensively replicate within macrophages. Through the work of genome-wide association studies (GWAS), there is growing evidence to suggest that the microbial imbalance between commensal and pathogenic bacteria in the gut is aided by a defect in the innate immune system. Autophagy represents a recently investigated pathway that is believed to contribute to the pathogenesis of CD, with studies identified a variant of the autophagy gene, ATG16L1, as a susceptibility gene. The aim of my thesis was to study the cellular and molecular mechanism promoted by E.coli strains in epithelial cells and to assess their contribution to IBD pathology. To achieve this we focused on developing both an in vitro and in vivo model of AIEC infection. This allowed us to further our knowledge on possible mechanisms utilised by AIEC that promoted their survival, as well as developing a better understanding of host reactions. We demonstrate a new survival mechanism promoted by E.coli HM605, whereby it induces the expression of the anti-apoptotic proteins Bcl-XL and BCL2, all of which is exacerbated in an autophagy deficient system. We have also demonstrated the presence of AIEC-induced inflammasome responses in epithelial cells which are exacerbated in an autophagy deficient system and expression of NOD-like receptors (NLRs) which might mediate inflammasome responses in vivo. Finally, we used the Citrobacter rodentium model of infectious colitis to identify Pellino3 as an important mediator in the NOD2 pathway and regulator of intestinal inflammation. In summary, we have developed robust and versatile models of AIEC infection as well as provide new insights into AIEC mediated survival pathways. The collected data provides a new perception into why AIEC bacteria are able to prosper in conditions associated with Crohn’s disease patients with a defect in autophagy.

Critical role of interleukin 5 and eosinophils in concanavalin A-induced hepatitis in mice.

BACKGROUND & AIMS: Eosinophils are observed in several liver diseases, but their contribution in the pathogenesis of these disorders remains poorly investigated. Concanavalin A (Con A)-induced hepatitis is an experimental model of immune-mediated liver injury in which natural killer T (NKT) cells play a critical role through the production of interleukin (IL)-4 and the expression of Fas ligand (FasL). Because activated NKT cells also produce IL-5, a critical cytokine for eosinophil maturation and function, the role of IL-5 was investigated in this model. METHODS: IL-5-deficient mice, eosinophil depletion in wild-type (WT) mice, and NKT cell transfer from WT- or IL-5-deficient mice into NKT cell-deficient mice were used to assess the role of IL-5 and eosinophils. RESULTS: Liver eosinophil infiltrate and IL-5 production were observed after Con A challenge. Liver injury was dramatically reduced in IL-5-deficient or eosinophil-depleted mice. In addition, residual hepatitis observed in Fas-deficient mice was abolished after IL-5 neutralization. Finally, we showed that NKT cells constituted a critical source of IL-5. Indeed, transfer of WT NKT cells to mice lacking NKT cells restored liver injury, whereas transfer of IL-5-deficient NKT cells did not. CONCLUSIONS: These observations highlight the pathologic role of IL-5 and eosinophils in experimental immune-mediated hepatitis.

The pathophysiology of eosinophilic esophagitis: Altered mechanisms of antigen detection in the esophagus

Recently, a chronic idiopathic disease of the esophagus has emerged, which is now known as eosinophilic esophagitis (EoE). Incomplete knowledge regarding the pathogenesis of EoE has limited treatment options. EoE is known to be a Th2-type immune-mediated disorder. Based on previous studies in both patients and experimental models, it is possible that an abnormal reaction to antigen mediates the pathophysiology of EoE. In this thesis, symptoms and signs unique to EoE were identified by an age-matched, case-controlled study of 326 patients with EoE and gastroesophageal reflux disease. The molecular mechanisms involved in antigen detection in the esophagus, in relation to EoE were then investigated. Esophageal epithelial cells were found, for the first time, to be capable of acting as non-professional antigen presenting cells, with the ability to engulf, process and present antigen on MHC class II to T helper lymphocytes. Antigen presentation by esophageal epithelial cells was induced by interferon-γ, which is increased in biopsies from patients with EoE. Next, it was discovered that esophageal epithelial cell lines expressed functional toll-like receptor (TLR) 2 and TLR3, but in esophageal mucosal biopsies only infiltrating immune cells (including eosinophils) expressed TLR2 and TLR3. Finally, the potential involvement of IgE in the pathogenesis of esophageal inflammation was investigated. IgE in the esophagus was found to be present on mast cells, which are increased in density in the esophageal mucosae of patients with EoE and especially those with a history of atopy. Mechanisms of antigen detection may mediate the pathophysiology of EoE in the esophagus through antigen presentation by epithelial cells, detection by TLRs on immune cells and detection through IgE on mucosal mast cells. Together, these findings demonstrate that mechanisms of antigen detection may actually contribute to the pathophysiology of EoE. Through increased understanding of the mechanisms of EoE, the results of this thesis may contribute to future therapy.