Charlotte Admyre

Exosomes with Immune Modulatory Features Are Present in Human Breast Milk

Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant’s immune system. Exosomes are nanovesicles (30–100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant’s immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-γ production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3+CD4+CD25+ T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.

Direct exosome stimulation of peripheral humanT cells detected by ELISPOT

Exosomes from APC are nano‐vesicles that can induce antigen‐specific T cell responses and are presently explored as therapeutic tools in different clinical settings. Investigations of the capacity of exosomes to stimulate T cells in vitro have mostly been performed on T cell hybridomas, clones or lines. Whether exosomes can stimulate T cells directly or need the presence of dendritic cells (DC) is debated. We could detect exosome‐induced antigen‐specific CD8+ T cell responses in peripheral blood from humans. Exosomes from monocyte‐derived DC (MDDC) were loaded with a mix of 23 immunogenic peptides from EBV, CMV and influenza virus, and added to autologous peripheral CD8+ T cells. IFN‐γ‐producing cells were detected by enzyme‐linked immunospot assay (ELISPOT). MDDC‐exosomes induced IFN‐γ production in CD8+ T cells without addition of DC. The response was exosome dose dependent, and dependent on exosomal MHC class I. Furthermore, we detected an enhanced T cell stimulatory capacity by exosomes from lipopolysaccharide‐matured MDDC compared to exosomes from immature MDDC. Exosomes could also induce TNF‐α production. These results show, for the first time, that exosomes can directly stimulate human peripheral CD8+ T cells in an antigen‐specific manner and that ELISPOT is a suitable method for detecting exosome‐induced peripheral T cell responses. This system may provide a useful tool when developing exosomes as therapeutic agents.

Exosomes – nanovesicles with possible roles in allergic inflammation

Exosomes are nano‐sized membrane vesicles which are released extracellularly after fusion of multivesicular endosomes with the cell membrane. Despite their characteristic composition of proteins compared to the cell membrane, no exosome‐specific molecule has so far been characterized. Exosomes are found in bronchoalveolar lavage (BAL), urine, serum and breast milk, and are released from several cells implicated in allergy including mast cells, dendritic cells (DC), T cells and epithelial cells. Antigen‐loaded exosomes have been shown to be highly immunogenic and we propose that exosomes could be a modulating factor in allergic responses. Allergen‐presenting exosomes could transport allergen and stimulate allergen‐specific T cells, and possibly also biasing T cell responses depending on the molecules present on the exosome surface. Furthermore, exosomes from mast cells, highly active in allergic reactions, have been found to induce DC maturation and also to be able to transport functional RNA to recipient cells, suggesting a new pathway for cell communication. Reversely, tolerizing exosomes e.g. tolerosomes, from gut or breast milk, could block an allergic response or prevent allergy development. A better understanding of the role of exosomes in allergies could make us understand how allergy can be prevented or lead to the development of more efficient treatments.

Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma

Leukotrienes (LTs) are potent pro‐inflammatory mediators involved in asthma. Exosomes, nanosized vesicles released from various cells, can stimulate or down‐regulate immune responses, depending on the state and nature of the originating cell. We have recently shown an altered exosome profile in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis, but their role in asthma is unknown. Our aims were to investigate whether exosomes from BALF have LT biosynthetic capacity and to explore phenotypic and functional characteristics of BALF exosomes in asthma.

Different types of in vitro generated human monocyte-derived dendritic cells release exosomes with distinct phenotypes

Human in vitro generated dendritic cells and the exosomes they release are potential tools for the modulation of immune responses. Here, we characterized differently generated monocyte‐derived dendritic cells (MDDCs) and their exosomes. Culturing of peripheral CD14+ cells from the same individuals with either interleukin (IL)‐4 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (conventional MDDCs) or alternatively with IL‐4 and IL‐3 generated immature MDDCs in 7 days. Fluorescence‐activated cell sorting (FACS) analysis showed that the IL‐4/IL‐3‐generated MDDCs had significantly lower percentages of CD1a+, CD40+ and CD80+ cells and a higher percentage of CD86+ cells as compared with conventional MDDCs. In addition, IL‐4/IL‐3‐generated MDDCs had significantly higher densities of major histocompatibility complex (MHC) class I [human leucocyte antigen (HLA)‐ABC], MHC class II (HLA‐DR), CD11c and the tetraspanin CD81 as compared with conventional MDDCs. In a comparison of their ability to stimulate CD8+ T cells, we found that the IL‐4/IL‐3 MDDCs were slightly more efficient than the conventional MDDCs at inducing interferon (IFN)‐γ release in response to viral peptides. Exosome morphology was confirmed by electron microscopy and exosome phenotypes were analysed by flow cytometry and western blot. In comparison to exosomes from conventional MDDCs, exosomes from IL‐4/IL‐3‐generated MDDCs showed significantly stronger signals for HLA‐ABC, HLA‐DR, CD11c, CD63 and CD81. Thus, phenotypically the exosomes largely reflected their MDDCs of origin. When exosomes were loaded with viral peptides, both types of exosomes induced IFN‐γ release from CD8+ T cells. Our findings might have significance for the development of DC‐ and exosome‐based therapies.

Differences in exosome populations in human breast milk in relation to allergic sensitization and lifestyle

Breast‐feeding has many beneficial effects on the developing immune system of the newborn. Breast milk contains immunoregulatory factors, such as nano‐sized vesicles named exosomes. This study aimed at characterizing breast milk exosomes from human early milk and mature milk and to investigate whether allergic sensitization and an anthroposophic lifestyle could influence the exosome profile.

Topical Treatment with the Toll-like Receptor Agonist DIMS0150 Has Potential for Lasting Relief of Symptoms in Patients with Chronic Active Ulcerative Colitis by Restoring Glucocorticoid Sensitivity

Background:Patients with chronic active ulcerative colitis (UC) are regarded as treatment failures and represent an area of high unmet medical need, as normally the only remaining option is colectomy. Methods:We treated a total of eight chronic active severe UC outpatients with the immunomodulatory agent DIMS0150 as an add-on to current therapies. Seven patients received a single topical dose of 30 mg and one special case subject received three doses with 4 weeks between dosing occasions. All patients were classed as treatment failures and were elected for colectomy. Efficacy evaluation was determined in terms of colitis activity index, endoscopic improvement, and histologic disease activity assessed primarily at week 12 with a follow-up period of over 2 years. Glucocorticoid sensitivity was assayed by in vitro measurement of interleukin 6. Results:All patients demonstrated a pronounced and rapid reduction in their colitis activity index within 1 week following a single intracolonic administration via colonoscope of the agent DIMS0150. Further improvements were evident at week 4, resulting in a clinical response rate for the single-dose treatment of 71%, with 43% in clinical remission. By week 12 the clinical response and remission rates had reached 82% and 71%, respectively. A follow-up period of over 2 years posttreatment indicated that all but one of the treated patients had avoided the need for colectomy, with the longest patient being in symptom-free remission for over 27 months. Treatment with DIMS0150 restored glucocorticoid sensitivity. Conclusions:DIMS0150 may have the potential to be an effective agent to treat chronic active UC patients with the prospect to avoid colectomy on a long-term basis and is currently the subject of a clinical phase III study (EudraCT number: 2011-003130-14).

Immunomodulatory oligonucleotides inhibit neutrophil migration by decreasing the surface expression of interleukin-8 and leukotriene B4 receptors.

Neutrophils play important roles in many inflammatory diseases. The migration of neutrophils to the inflammatory site is tightly regulated by specific chemokines, of which interleukin‐8 (IL‐8) and leukotriene B4 (LTB4) constitute key mediators by binding to the surface receptors CXCR1/2 and BLT1, respectively. Oligonucleotides (ODN) containing CpG motifs mediate potent immunomodulatory effects through binding to Toll‐like receptor 9. So far, knowledge on how ODN can affect neutrophil migration during inflammation is lacking. This study demonstrates that several novel CpG ODN significantly down‐regulate the surface expression of CXCR1/2 and BLT1. In addition, the ODN significantly blocked IL‐8‐induced and LTB4‐induced neutrophil migration in vitro, as well as leucocyte migration in vivo demonstrated in mice by intravital microscopy and in a model of airway inflammation. The down‐regulation of CXCR1 is rapid, occurring 15 min after ODN stimulation, and can be mediated through an endosomally independent mechanism. Inhibition of the IL‐8 and LTB4 pathways may provide new opportunities of therapeutic intervention using ODN to reduce neutrophil infiltration during inflammation.

Treatment of Ulcerative Colitis Patients by Local Application of the Toll likeReceptor-9 Agonist DIMS0150

Background and aim: The Toll like receptor 9 (TLR9) has over the years received growing interest as a potential point of therapeutic intervention in the treatment of ulcerative colitis. The aim of this study was to evaluate a TLR9 agonist DIMS0150 as a new treatment option for patients with ulcerative colitis. Methods: A randomized, double-blind, placebo controlled, multicenter trial was conducted in ulcerative colitis patients with moderate to severe disease activity despite concomitant steroid medication. 34 patients were randomized to receive a single rectal administration of 30 mg of DIMS0150 or placebo. Results: Clinical response at week 4 was 33% and 41% in the placebo and DIMS0150 treated group, respectively. The proportion of patients in clinical remission at week 4 was 13%, observed only in the DIMS0150 group. Likewise, histological response or remission at week 4 was 27% (p-value 0.06) in the DIMS0150 group only. Rates of sustained clinical response were 27% (p-value 0.06) and 31%, in the DIMS0150 treated group across week 4 and 3 months respectively. Sub score analysis of the disease activity indices indicated a highly statistical difference at week 4 from baseline only in the DIMS0150 treated group for all 4 DAI sub scores. Conclusion: Although statistical significant results for the primary endpoints could not be obtained in this small study, DIMS0150 treatment showed positive signals suggesting that TLR-9 activation could be an interesting upcoming therapeutic option.

Clinical efficacy of the Toll-like receptor 9 agonist cobitolimod using patient-reported-outcomes defined clinical endpoints in patients with ulcerative colitis

BACKGROUND The Toll-like-receptor 9 (TLR-9) agonist cobitolimod (DIMS0150, Kappaproct®) is a promising therapeutic option for ulcerative colitis (UC) patients. AIMS The objectives of this post-hoc analysis using the COLLECT study data was to investigate the clinical effects of cobitolimod using patient-reported-outcomes (PRO) defined endpoints. METHODS Dual topical administration of cobitolimod was studied in a randomised, multicentre clinical trial named COLLECT in moderate-to-severe UC patients. Symptomatic remission (SR) was studied in 104 patients based on their e-diary records and was defined as absence of blood in stool and a mean daily stool frequency (SF) < 4. RESULTS SR was achieved at week 4 in 17.1% of cobitolimod vs. 5.9% of placebo treated patients (p = 0.13), at week 8 in 35.7% vs. 17.6% (p = 0.07), and at week 12 in 38.6% vs. 17.6% (p = 0.04) of the patients, respectively. SR rates with cobitolimod and placebo in anti-TNFα experienced patients were smaller but with a broadly similar relative effect-size to anti-TNFα naïve patients. Clinical efficacy was higher in patients with moderate compared to severe disease. CONCLUSIONS Application of the Toll-like-receptor 9 (TLR-9) agonist cobitolimod is able to induce remission as assessed by PRO measures in UC patients with moderate-to-severe activity as well as in anti-TNFα experienced and naïve patients supporting the overall efficacy of the substance.