Distinct populations of eosinophils in the human thymus with capacity to modulate thymocyte maturation.

Local differentiation of eosinophil precursors occurs in the human thymus. Thymic eosinophils are often positioned in the corticomedullary junction between the CD4+ CD8+ double-positive (DP) thymocytes and the CD4+ or CD8+ single-positive (SP) thymocytes. The aims of this study were to (1) determine if there are distinct thymic eosinophil populations that differ from the blood eosinophil populations and (2) evaluate the capacity of thymic eosinophils to promote the development of SP thymocytes from DP thymocytes. Thymic and blood eosinophils from thymectomized infants (n = 7) were compared regarding the expression of 34 molecules using cytometry by time-of-flight (CyTOF). In addition, FACS-sorted thymic eosinophils were co-cultured with autologous CD3/CD28-stimulated DP, CD4 SP, and CD8 SP thymocytes and analysed by flow cytometry and CyTOF. X-shift clustering analysis and viSNE dimensionality reduction were performed. Seven eosinophil populations were identified within the blood and thymus, respectively, five of which were specific for either tissue. Whereas the blood eosinophil populations varied between individuals, the thymic eosinophil populations were more uniform. The eosinophil-thymocyte co-cultures resulted in (1) an increase in CD4 SP thymocytes when eosinophils were cultured with DP thymocytes, (2) decreased frequency of CD8 SP thymocytes when these were cultured with eosinophils, and (3) a more mature thymic phenotype when eosinophils were cultured with CD4 SP thymocytes. Thymic eosinophils are a specialized population of eosinophils with a distinct phenotype that separates them from their blood counterparts, and in vitro they appear to favour CD4 SP thymocyte development to the detriment of CD8 SP thymocytes.

Long-Term Follow-Up of Newborns with 22q11 Deletion Syndrome and Low TRECs.

BACKGROUND: Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). PURPOSE: To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. METHODS: Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. RESULTS: At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. CONCLUSIONS: This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. CLINICAL IMPLICATIONS: This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices.

Eosinophils interact with thymocytes and proliferate in the human thymus.

Eosinophils differentiate and mature in the thymus, outside of the bone marrow, in healthy individuals. Locally developed thymic eosinophils may contribute to the maturation and selection of human thymocytes.

Altered expression of autoimmune regulator in infant down syndrome thymus, a possible contributor to an autoimmune phenotype.

Down syndrome (DS), caused by trisomy of chromosome 21, is associated with immunological dysfunctions such as increased frequency of infections and autoimmune diseases. Patients with DS share clinical features, such as autoimmune manifestations and specific autoantibodies, with patients affected by autoimmune polyendocrine syndrome type 1. Autoimmune polyendocrine syndrome type 1 is caused by mutations in the autoimmune regulator (AIRE) gene, located on chromosome 21, which regulates the expression of tissue-restricted Ags (TRAs) in thymic epithelial cells. We investigated the expression of AIRE and TRAs in DS and control thymic tissue using quantitative PCR. AIRE mRNA levels were elevated in thymic tissue from DS patients, and trends toward increased expression of the AIRE-controlled genes INSULIN and CHRNA1 were found. Immunohistochemical stainings showed altered cell composition and architecture of the thymic medulla in DS individuals with increased frequencies of AIRE-positive medullary epithelial cells and CD11c-positive dendritic cells as well as enlarged Hassall's corpuscles. In addition, we evaluated the proteomic profile of thymic exosomes in DS individuals and controls. DS exosomes carried a broader protein pool and also a larger pool of unique TRAs compared with control exosomes. In conclusion, the increased AIRE gene dose in DS could contribute to an autoimmune phenotype through multiple AIRE-mediated effects on homeostasis and function of thymic epithelial cells that affect thymic selection processes.

Constitutively low expression of collagen XIII alpha 1 may help explain the vulnerability of the inferior rectus muscle to thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) has a predilection for inferior rectus muscle that has never been explained. We conducted immunohistochemical staining for the soluble cleaved form of collagen XIII alpha 1 (COL13A1) and found constitutively low expression of COL13A1 in normal human inferior rectus muscles and moderate expression of COL13A1 in normal human medial rectus muscles. COL13A1 is known to be essential to development and maintenance of neuromuscular junctions and there is some evidence to suggest it may help support normal immune function. The combination of constitutively low expression of COL13A1, high physiological and metabolic demands, and consequentially relatively high exposure to stressors via the blood stream may help explain the particular vulnerability of inferior rectus to TAO compared to other extraocular muscles.

Overexpression of collagen XIII in extraocular fat affected by active thyroid-associated ophthalmopathy: A crucial piece of the puzzle?

Thyroid-associated ophthalmopathy (TAO) causes irreversible increase in extraocular fat volume that contributes to the risk of exophthalmos and compressive optic neuropathy. Collagen XIII is implicated in uncontrolled cell growth in some tumours, but we are not aware of any studies of collagen XIII in TAO-affected solid tissue to date. We conducted immunohistochemical staining for collagen XIII alpha 1 (COL13A1), present in both the transmembrane and cleaved forms of collagen XIII, in consecutive prospectively collected human extraocular tissue specimens from patients with TAO and controls. We identified overexpression of collagen XIII in active TAO-affected fat. We discuss how species and cell-type specific responses of collagen XIII to stressors may help explain the different phenotypes of TAO.

Human thymic epithelial primary cells produce exosomes carrying tissue-restricted antigens.

Exosomes are nano-sized vesicles released by cells into the extracellular space and have been shown to be present in thymic tissue both in mice and in humans. The source of thymic exosomes is however still an enigma and hence it is not known whether thymic epithelial cells (TECs) are able to produce exosomes. In this work, we have cultured human TECs and isolated exosomes. These exosomes carry tissue-restricted antigens (TRAs), for example, myelin basic protein and desmoglein 3. The presence of TRAs indicates a possible role for thymic epithelium-derived exosomes in the selection process of thymocytes. The key contribution of these exosomes could be to disseminate self-antigens from the thymic epithelia, thus making them more accessible to the pool of maturing thymocytes. This would increase the coverage of TRAs within the thymus, and facilitate the process of positive and negative selection.

Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes.

Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell's plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.

Immunophenotype in orofacial granulomatosis with and without Crohn's disease.

OBJECTIVES: The aim of this investigation was to characterise and compare the inflammatory infiltrates in patients with orofacial granulomatosis solely (OFG-S) and OFG with coexisting Crohn's disease (OFG+CD). STUDY DESIGN: Biopsy specimens with granulomas were obtained from patients with OFG-S (n=11) and OFG+CD (n=11) and immunostained with antibodies against CD1a, CD3, CD4, CD8, CD11c, CD20, CD68 and mast cell tryptase, followed by quantitative analysis. RESULTS: Analyses of the connective tissue revealed a significantly higher number of CD3-expressing T cells and CD11c-expressing dendritic cells in the connective tissue of patients with OFG-S compared to patients with OFG+CD. Mast cells displayed a high level of activation, although no significant difference was detected when comparing the two groups. CONCLUSIONS: The results show a different composition of the inflammatory infiltrate in patients with OFG-S compared to patients with OFG+CD. The present observations support that partly-divergent immune mechanisms are involved in these two different subcategories of OFG.

Neonatal exposure to staphylococcal superantigen improves induction of oral tolerance in a mouse model of airway allergy.

The hygiene hypothesis suggests that lack of microbial stimulation in early infancy may lead to allergy, but it has been difficult to identify particular protective microbial exposures. We have observed that infants colonised in the first week(s) of life with Staphylococcus aureus have lower risk of developing food allergy. As many S. aureus strains produce superantigens with T-cell stimulating properties, we here investigate whether neonatal mucosal exposure to superantigen could influence the capacity to develop oral tolerance and reduce sensitisation and allergy. BALB/c mice were exposed to staphylococcal enterotoxin A (SEA) as neonates and fed with OVA as adults, prior to sensitisation and i.n. OVA challenge. Our results show that SEA pre-treated mice are more efficiently tolerised by OVA feeding, as shown by lower lung-cell infiltration and antigen-specific IgE response in the SEA pre-treated mice, compared with sham-treated mice. This was not due to deletion or anergy of lymphocytes by SEA treatment, because the SEA pre-treated mice that were fed with PBS showed similar inflammatory response as the sham-treated PBS-fed mice. Our results suggest that strong T-cell activation in infancy conditions the mucosal immune system and promotes development of oral tolerance.

Galectin-3 functions as an opsonin and enhances the macrophage clearance of apoptotic neutrophils.

Galectin-3, a beta-galactoside binding, endogenous lectin, takes part in various inflammatory events and is produced in substantial amounts at inflammatory foci. We investigated whether extracellular galectin-3 could participate in the phagocytic clearance of apoptotic neutrophils by macrophages, a process of crucial importance for termination of acute inflammation. Using human leukocytes, we show that exogenously added galectin-3 increased the uptake of apoptotic neutrophils by monocyte-derived macrophages (MDM). Both the proportion of MDM that engulfed apoptotic prey and the number of apoptotic neutrophils that each MDM engulfed were enhanced in the presence of galectin-3. The effect was lactose-inhibitable and required galectin-3 affinity for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins, since a mutant lacking this activity was without effect. The enhanced uptake relied on the presence of galectin-3 during the cellular interaction and was paralleled by lectin binding to apoptotic cells as well as MDM in a lactose-dependent manner. These findings suggest that galectin-3 functions as a bridging molecule between phagocyte and apoptotic prey, acting as an opsonin. The process of clearance, whereby apoptotic neutrophils are removed by macrophages, is crucial for the resolution of acute inflammation and our data imply that the increased levels of galectin-3 often found at inflammatory sites could potently affect this process.

Serum-derived exosomes from antigen-fed mice prevent allergic sensitization in a model of allergic asthma.

Oral tolerance is an active process that starts with sampling of luminal antigens by the intestinal epithelial cells (IEC), followed by processing and assembly with major histocompatibility complex class II and subsequently a release of tolerogenic exosomes (tolerosomes) from the IEC. We have previously shown that tolerosomes can be isolated from serum shortly after an antigen feed, and will potently transfer antigen-specific tolerance to naive recipients. Here we study the capacity of the tolerosomes to protect against allergic sensitization in a mouse model of allergic asthma. Serum or isolated serum exosomes from tolerized BALB/c donor mice were transferred to syngeneic recipients followed by sensitization and intranasal exposure to ovalbumin (OVA). Blood, bronchoalveolar lavage (BAL) and lymph nodes were sampled 24 hr after the final exposure. The number of eosinophils was counted in BAL fluid and the levels of immunoglobulin E (IgE) and OVA-specific IgE were measured in serum. Mediastinal and coeliac lymph nodes were analysed by flow cytometry. The animals receiving serum from OVA-fed mice displayed significantly lower numbers of airway eosinophils and lower serum levels of total IgE as well as of OVA-specific IgE compared with controls. Moreover, the tolerant animals showed a significantly higher frequency of activated T cells with a regulatory phenotype in both mediastinal and coeliac lymph nodes. The results show that serum or isolated serum exosomes obtained from OVA-fed mice and administered intraperitoneally to naive recipient mice abrogated allergic sensitization in the recipients.

MHC expression and chemokine production in the murine vagina following intra-vaginal administration of ligands to toll-like receptors 3, 7 and 9.

The expression of MHC class I, MHC class II and the chemokines IP-10, MIP-1alpha, RANTES, fractalkine and I-TAC has been analyzed after intra-vaginal treatment with three synthetic toll-like receptors (TLR) agonists-double-stranded RNA (poly I:C), imiquimod and CpG-rich oligonucleotides (CpG-ODN). These compounds act mainly through TLR3, TLR7 and TLR9, respectively. CpG-ODN induced an accumulation of leucocytes in the vagina, and a strong up-regulation of MHC class I expression on both leucocytes and epithelial cells. Imiquimod and poly I:C induced a weak MHC class I up-regulation in the epithelium but not in the lamina propria. Neither treatment had any profound effect on expression of MHC class II on epithelial cells but poly I:C and to a lesser extent CpG-ODN, up-regulated MHC class II staining intensity which, in the case of CpG-ODN, treatment, was associated with a strong accumulation of CD11c-positive dendritic cells. All three treatments induced an early (8h) but transient IP-10 response. Imiquimod and CpG-ODN, but not poly I:C induced an early MIP-1alpha response which remained for at least 7 days in CpG-ODN-treated animals but not in imiquimod-treated mice. Poly I:C and CpG-ODN, but not imiquimod, induced significant levels of RANTES at different time-points post-treatment. None of the treatments induced any significant changes in the levels of fractalkine, I-TAC or IFN-alpha. These studies have implications for the manipulation of the genital immune response and also improving the outcome of vaginal immunotherapy.

IFN type I and II induce BAFF secretion from human decidual stromal cells.

B cell activating factor (BAFF) is a critical cytokine for maturation of immature B cells. In murine lymph nodes, BAFF is mainly produced by podoplanin-expressing stromal cells. We have previously shown that circulating BAFF levels are maximal at birth, and that farmers' children exhibit higher BAFF levels in cord blood than non-farmers' children. Here, we sought to investigate whether maternal-derived decidual stromal cells from placenta secrete BAFF and examine what factors could stimulate this production. We found that podoplanin is expressed in decidua basalis and in the underlying villous tissue as well as on isolated maternal-derived decidual stromal cells. Decidual stromal cells produced BAFF when stimulated with IFN-γ and IFN-α, and NK cells and NK-T-like cells competent of IFN-γ production were isolated from the decidua. Finally, B cells at different maturational stages are present in decidua and all expressed BAFF-R, while stromal cells did not. These findings suggest that decidual stromal cells are a cellular source of BAFF for B cells present in decidua during pregnancy.

Delivery of Small Interfering RNAs to Cells via Exosomes.

Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot.

Thymic exosomes promote the final maturation of thymocytes.

Extensive knowledge has been gained the last years concerning mechanisms underlying the selection of single positive thymocytes in the thymic medulla. Less is known regarding other important processes in the thymic medulla such as the regulation of late stage thymocyte maturation. We have previously reported that exosomes are abundant in the thymus with a phenotype that indicates an epithelial cell origin and immunoregulatory properties. In this study we use an in vitro system to investigate the effects of thymic exosomes on the maturation of single positive thymocytes as well as effects on nTreg formation. We show that thymic exosomes promote the maturation of single positive CD4+CD25- cells into mature thymocytes with S1P1+Qa2+ and CCR7+Qa2+ phenotypes. Furthermore, we show that thymic exosomes reduce the formation of CD4+CD25+FoxP3+ thymocytes and that these exosome effects are independent of dendritic cell co-stimulation but require intact exosomal RNA content and surface proteins. An efficient direct uptake of exosomes by both thymocytes and thymic DC's is also demonstrated. In conclusion, this study demonstrates that exosomes may represent a new route of communication within the thymus.