Camilla Janefjord

Lysosomal Network Proteins as Potential Novel CSF Biomarkers for Alzheimer’s Disease

Abstract The success of future intervention strategies for Alzheimer’s disease (AD) will likely rely on the development of treatments starting early in the disease course, before irreversible brain damage occurs. The pre-symptomatic stage of AD occurs at least one decade before the clinical onset, highlighting the need for validated biomarkers that reflect this early period. Reliable biomarkers for AD are also needed in research and clinics for diagnosis, patient stratification, clinical trials, monitoring of disease progression and the development of new treatments. Changes in the lysosomal network, i.e., the endosomal, lysosomal and autophagy systems, are among the first alterations observed in an AD brain. In this study, we performed a targeted search for lysosomal network proteins in human cerebrospinal fluid (CSF). Thirty-four proteins were investigated, and six of them, early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were significantly increased in the CSF from AD patients compared with neurological controls. These results were confirmed in a validation cohort of CSF samples, and patients with no neurochemical evidence of AD, apart from increased total-tau, were found to have EEA1 levels corresponding to the increased total-tau levels. These findings indicate that increased levels of LAMP-1, LAMP-2, LC3, Rab3 and Rab7 in the CSF might be specific for AD, and increased EEA1 levels may be a sign of general neurodegeneration. These six lysosomal network proteins are potential AD biomarkers and may be used to investigate lysosomal involvement in AD pathogenesis.

Decreased up-regulation of the interleukin-12Rβ2-chain and interferon-γ secretion and increased number of forkhead box P3-expressing cells in patients with a history of chronic Lyme borreliosis compared with asymptomatic Borrelia-exposed individuals

Lyme borreliosis (LB) can, despite adequate antibiotic treatment, develop into a chronic condition with persisting symptoms such as musculoskeletal pain, subjective alteration of cognition and fatigue. The mechanism behind this is unclear, but it has been postulated that an aberrant immunological response might be the cause. In this study we investigated the expression of the T helper 1 (Th1) marker interleukin (IL)‐12Rβ2, the marker for T regulatory cells, forkhead box P3 (FoxP3) and the cytokine profile in patients with a history of chronic LB, subacute LB, previously Borrelia‐exposed asymptomatic individuals and healthy controls. Fifty‐four individuals (12 chronic LB, 14 subacute LB, 14 asymptomatic individuals and 14 healthy controls) were included in the study and provided a blood sample. Mononuclear cells were separated from the blood and stimulated with antigens. The IL‐12Rβ2 and FoxP3 mRNA expression was analysed with real‐time reverse transcription–polymerase chain reaction (RT–PCR). The protein expression of IL‐12Rβ2 on CD3+, CD4+, CD8+ and CD56+ cells was assessed by flow cytometry. Furthermore, the secretion of interferon (IFN)‐γ, IL‐4, IL‐5, IL‐10, IL‐12p70 and IL‐13 was analysed by enzyme‐linked immunospot (ELISPOT) and/or enzyme‐linked immunosorbent assay (ELISA). Chronic LB patients displayed a lower expression of Borrelia‐specific IL‐12Rβ2 on CD8+ cells and also a lower number of Borrelia‐specific IFN‐γ‐secreting cells compared to asymptomatic individuals. Furthermore, chronic LB patients had higher amounts of Borrelia‐specific FoxP3 mRNA than healthy controls. We speculate that this may indicate that a strong Th1 response is of importance for a positive outcome of a Borrelia infection. In addition, regulatory T cells might also play a role, by immunosuppression, in the development of chronic LB.

PHA‐induced IL‐12Rβ2 mRNA expression in atopic and non‐atopic children

Background IL‐12 is a strong inducer of Th1 responses. Stimulation via the CD2 receptor increases IFN‐γ production and enhances the responsiveness of activated T‐cells to IL‐12, possibly due to an up‐regulation of the signal transducing β2 chain of the IL‐12 receptor (IL‐12Rβ2). Atopic children have a reduced Th1‐like immunity and a reduced CD2 expression. Our hypothesis is that atopic individuals have a reduced function of the CD2 pathway, causing reduced responsiveness to IL‐12 and decreased IFN‐γ production.

Protective properties of lysozyme on β-amyloid pathology: implications for Alzheimer disease.

The hallmarks of Alzheimer disease are amyloid-β plaques and neurofibrillary tangles accompanied by signs of neuroinflammation. Lysozyme is a major player in the innate immune system and has recently been shown to prevent the aggregation of amyloid-β1-40 in vitro. In this study we found that patients with Alzheimer disease have increased lysozyme levels in the cerebrospinal fluid and lysozyme co-localized with amyloid-β in plaques. In Drosophila neuronal co-expression of lysozyme and amyloid-β1-42 reduced the formation of soluble and insoluble amyloid-β species, prolonged survival and improved the activity of amyloid-β1-42 transgenic flies. This suggests that lysozyme levels rise in Alzheimer disease as a compensatory response to amyloid-β increases and aggregation. In support of this, in vitro aggregation assays revealed that lysozyme associates with amyloid-β1-42 and alters its aggregation pathway to counteract the formation of toxic amyloid-β species. Overall, these studies establish a protective role for lysozyme against amyloid-β associated toxicities and identify increased lysozyme in patients with Alzheimer disease. Therefore, lysozyme has potential as a new biomarker as well as a therapeutic target for Alzheimer disease.

The Placental Immune Milieu is Characterized by a Th2- and Anti-Inflammatory Transcription Profile, Regardless of Maternal Allergy, and Associates with Neonatal Immunity

How maternal allergy affects the systemic and local immunological environment during pregnancy and the immune development of the offspring is unclear.

Reduced IL-2-induced IL-12 responsiveness in atopic children

Atopy may be associated with a reduced T‐cell function particularly regarding maturation of T helper 1 (Th1) responses. We hypothesized that atopic children may have a reduced capacity to up‐regulate the β2 subunit of the interleukin‐12 (IL‐12) receptor (IL‐12Rβ2, the signal‐transducing component). The study included 38 children followed from birth to the age of 7 years. Twenty one had a cumulative history of atopic disease, whereas 17 had none. Sixteen out of 21 children also had atopic symptoms within the past year (current), out of whom 10 children had atopic airway symptoms. The expression of IL‐12Rβ2 mRNA was analyzed by quantitative real‐time PCR and the secretion of interferon‐γ (IFN‐γ), IL‐5 and IL‐10 was assessed by enzyme‐linked immunosorbent assay (ELISA). Children with current atopic airway symptoms and high levels of total IgE up‐regulated IL‐12Rβ2 mRNA expression less than non‐atopic children with low IgE levels after IL‐2 stimulation. This was accompanied by a low IL‐2‐ and IL‐12‐induced IFN‐γ production, possibly reflecting the reduced capacity of atopic children to up‐regulate the IL‐12 receptor. As IL‐2 is needed to initiate and sustain immune responses and IL‐12 promotes Th1 responses, this may contribute to the Th2‐skewed pattern in atopic children.

Beneficial effects of increased lysozyme levels in Alzheimer’s disease modelled in Drosophila melanogaster

Genetic polymorphisms of immune genes that associate with higher risk to develop Alzheimers disease (AD) have led to an increased research interest on the involvement of the immune system in AD pathogenesis. A link between amyloid pathology and immune gene expression was suggested in a genome‐wide gene expression study of transgenic amyloid mouse models. In this study, the gene expression of lysozyme, a major player in the innate immune system, was found to be increased in a comparable pattern as the amyloid pathology developed in transgenic mouse models of AD. A similar pattern was seen at protein levels of lysozyme in human AD brain and CSF, but this lysozyme pattern was not seen in a tau transgenic mouse model. Lysozyme was demonstrated to be beneficial for different Drosophila melanogaster models of AD. In flies that expressed Aβ1‐42 or AβPP together with BACE1 in the eyes, the rough eye phenotype indicative of toxicity was completely rescued by coexpression of lysozyme. In Drosophila flies bearing the Aβ1‐42 variant with the Arctic gene mutation, lysozyme increased the fly survival and decreased locomotor dysfunction dose dependently. An interaction between lysozyme and Aβ1‐42 in the Drosophila eye was discovered. We propose that the increased levels of lysozyme, seen in mouse models of AD and in human AD cases, were triggered by Aβ1‐42 and caused a beneficial effect by binding of lysozyme to toxic species of Aβ1‐42, which prevented these from exerting their toxic effects. These results emphasize the possibility of lysozyme as biomarker and therapeutic target for AD.

Pregnancy modulates the allergen-induced cytokine production differently in allergic and non-allergic women

The immunological environment during pregnancy may differ between allergic and non‐allergic women. This study investigates the effect of maternal allergy on the allergen‐induced cytokine and chemokine levels and whether pregnancy modulates these immune responses differently in allergic and non‐allergic women.