Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood

Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2(+) CD25(lo) NK cells were the predominant subset of NK cells within the blood. In contrast, CD2(−) CD25(hi) NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2(−) NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2(−) subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection

Nanovesicles from Malassezia sympodialis and Host Exosomes Induce Cytokine Responses – Novel Mechanisms for Host-Microbe Interactions in Atopic Eczema

BACKGROUND: Intercellular communication can occur via the release of membrane vesicles. Exosomes are nanovesicles released from the endosomal compartment of cells. Depending on their cell of origin and their cargo they can exert different immunoregulatory functions. Recently, fungi were found to produce extracellular vesicles that can influence host-microbe interactions. The yeast Malassezia sympodialis which belongs to our normal cutaneous microbial flora elicits specific IgE- and T-cell reactivity in approximately 50% of adult patients with atopic eczema (AE). Whether exosomes or other vesicles contribute to the inflammation has not yet been investigated. OBJECTIVE: To investigate if M. sympodialis can release nanovesicles and whether they or endogenous exosomes can activate PBMC from AE patients sensitized to M. sympodialis. METHODS: Extracellular nanovesicles isolated from M. sympodialis, co-cultures of M. sympodialis and dendritic cells, and from plasma of patients with AE and healthy controls (HC) were characterised using flow cytometry, sucrose gradient centrifugation, Western blot and electron microscopy. Their ability to stimulate IL-4 and TNF-alpha responses in autologous CD14, CD34 depleted PBMC was determined using ELISPOT and ELISA, respectively. RESULTS: We show for the first time that M. sympodialis releases extracellular vesicles carrying allergen. These vesicles can induce IL-4 and TNF-α responses with a significantly higher IL-4 production in patients compared to HC. Exosomes from dendritic cell and M. sympodialis co-cultures induced IL-4 and TNF-α responses in autologous CD14, CD34 depleted PBMC of AE patients and HC while plasma exosomes induced TNF-α but not IL-4 in undepleted PBMC. CONCLUSIONS: Extracellular vesicles from M. sympodialis, dendritic cells and plasma can contribute to cytokine responses in CD14, CD34 depleted and undepleted PBMC of AE patients and HC. These novel observations have implications for understanding host-microbe interactions in the pathogenesis of AE

A CD8+ T cell transcription signature predicts prognosis in autoimmune disease.

Autoimmune diseases are common and debilitating, but their severe manifestations could be reduced if biomarkers were available to allow individual tailoring of potentially toxic immunosuppressive therapy. Gene expression-based biomarkers facilitating such tailoring of chemotherapy in cancer, but not autoimmunity, have been identified and translated into clinical practice. We show that transcriptional profiling of purified CD8(+) T cells, which avoids the confounding influences of unseparated cells, identifies two distinct subject subgroups predicting long-term prognosis in two autoimmune diseases, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), a chronic, severe disease characterized by inflammation of medium-sized and small blood vessels, and systemic lupus erythematosus (SLE), characterized by autoantibodies, immune complex deposition and diverse clinical manifestations ranging from glomerulonephritis to neurological dysfunction. We show that the subset of genes defining the poor prognostic group is enriched for genes involved in the interleukin-7 receptor (IL-7R) pathway and T cell receptor (TCR) signaling and those expressed by memory T cells. Furthermore, the poor prognostic group is associated with an expanded CD8(+) T cell memory population. These subgroups, which are also found in the normal population and can be identified by measuring expression of only three genes, raise the prospect of individualized therapy and suggest new potential therapeutic targets in autoimmunity

Murine Dendritic Cells Transcriptional Modulation upon Paracoccidioides brasiliensis Infection

Limited information is available regarding the modulation of genes involved in the innate host response to Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis. Therefore, we sought to characterize, for the first time, the transcriptional profile of murine bone marrow-derived dendritic cells (DCs) at an early stage following their initial interaction with P. brasiliensis. DCs connect innate and adaptive immunity by recognizing invading pathogens and determining the type of effector T-cell that mediates an immune response. Gene expression profiles were analyzed using microarray and validated using real-time RT-PCR and protein secretion studies. A total of 299 genes were differentially expressed, many of which are involved in immunity, signal transduction, transcription and apoptosis. Genes encoding the cytokines IL-12 and TNF-α, along with the chemokines CCL22, CCL27 and CXCL10, were up-regulated, suggesting that P. brasiliensis induces a potent proinflammatory response in DCs. In contrast, pattern recognition receptor (PRR)-encoding genes, particularly those related to Toll-like receptors, were down-regulated or unchanged. This result prompted us to evaluate the expression profiles of dectin-1 and mannose receptor, two other important fungal PRRs that were not included in the microarray target cDNA sequences. Unlike the mannose receptor, the dectin-1 receptor gene was significantly induced, suggesting that this β-glucan receptor participates in the recognition of P. brasiliensis. We also used a receptor inhibition assay to evaluate the roles of these receptors in coordinating the expression of several immune-related genes in DCs upon fungal exposure. Altogether, our results provide an initial characterization of early host responses to P. brasiliensis and a basis for better understanding the infectious process of this important neglected pathogen

Phosphatidylserine Targets Single-Walled Carbon Nanotubes to Professional Phagocytes In Vitro and In Vivo

Broad applications of single-walled carbon nanotubes (SWCNT) dictate the necessity to better understand their health effects. Poor recognition of non-functionalized SWCNT by phagocytes is prohibitive towards controlling their biological action. We report that SWCNT coating with a phospholipid “eat-me” signal, phosphatidylserine (PS), makes them recognizable in vitro by different phagocytic cells - murine RAW264.7 macrophages, primary monocyte-derived human macrophages, dendritic cells, and rat brain microglia. Macrophage uptake of PS-coated nanotubes was suppressed by the PS-binding protein, Annexin V, and endocytosis inhibitors, and changed the pattern of pro- and anti-inflammatory cytokine secretion. Loading of PS-coated SWCNT with pro-apoptotic cargo (cytochrome c) allowed for the targeted killing of RAW264.7 macrophages. In vivo aspiration of PS-coated SWCNT stimulated their uptake by lung alveolar macrophages in mice. Thus, PS-coating can be utilized for targeted delivery of SWCNT with specified cargoes into professional phagocytes, hence for therapeutic regulation of specific populations of immune-competent cells

Activation of human dendritic cells : role of the allergenic yeast Malassezia and NK cells

Immature bone marrow derived dendritic cells (DCs) patrol the periphery of our body, where they encounter and take up foreign antigens. Once activated, DCs migrate to regional lymph nodes to present peptides derived from these antigens to naive T cells, and elicit different T helper (Th) cell responses. One of the keys to understanding DC function is the investigation of their activation and maturation, and the process by which activated DCs can instruct the adaptive immune system to use appropriate defense mechanisms. Few studies have so far focused on the effect of yeast on DCs. The yeast Malassezia is part of the normal flora of our skin, but can act as an allergen and elicit specific IgE antibodies and T cell reactivity in patients with atopic eczema/dermatitis syndrome (AEDS). AEDS is a chronic inflammatory skin disease that is increasing in prevalence worldwide. Though, the mechanisms that determine whether an exposed individual becomes sensitized are still poorly understood. The interaction between genetic predisposition and environmental factors seems to be crucial. Little is also known about the modulation of DC function by other cells. Only recently it became evident that NK cells might play a role in affecting DC maturation and function. It is not known, however, if this interaction takes place in vivo, and if NK-DC interaction is affected by allergen exposure. We showed, by flow cytometry and confocal microscopy, that immature human DCs bind and internalize Malassezia yeast cells and allergenic components from the yeast. This ability of the DCs is lost upon maturation. Furthermore, we showed a mannose receptor-mediated uptake of some yeast components. Using time-lapse photography we followed the kinetics of the uptake of Malassezia, and found that the yeast is taken up within 1 hour. Malassezia induces DC maturation (expression of CD80, CD83 and CD86), and production of cytokines like TNF-alpha and IL1beta, but not production of the Th1-inducing cytokine IL-12. Production of IL-18 increases after interaction with Malassezia, a phenomenon that might contribute to Th2 responses seen in AEDS patients. Finally, functional studies demonstrated that DCs pre-incubated with Malassezia induce proliferation in autologous peripheral blood mononuclear cells. Next we studied the distribution of NK cells in the skin of AEDS patients with particular emphasis on possible NK cell-DC interactions. Confocal microscopy revealed a few scattered NK (CD56+/CD3-) cells in the dermis of healthy individuals and in non-lesional skin from AEDS patients. However, NK cells were found to be differently distributed in lesional and Malassezia atopy patch test-positive skin, and for the first time, NK cell-DC contact in vivo was shown. By in vitro studies, we showed that DCs pre-stimulated with Malassezia are less susceptible to NK cell-induced cell death (51Cr release assay), suggesting that Malassezia affect the interaction through induction of DC maturation and production of soluble factors. Additionally, the results imply that the yeast has a direct effect on the NK cell cytotoxicity. We also showed that NK cells induce CD48 expression on DCs. The proportion of CD48+ cells increases in immature DCs, and even more so in those stimulated with Malassezia extract. The increased expression of CD48 on the DCs might be important for their ability to stimulate T cells, Besides, after contact with NK cells, the expression of CD83 and CD86 increases significantly in the Malassezia extract-stimulated DCs. IL-8 was shown to be induced in the NK cell-DC co-cultures. Taken together, the uptake of Malassezia and allergenic components from the yeast in the absence of IgE, imply that sensitization to Malassezia can be mediated by immature DCs in the skin. After interaction with Malassezia yeast cells, DCs become functionally mature and produce cytokines that might give rise to a Th2-like immune response. Our findings indicate that NK cells and DCs can interact in the skin, suggesting that NK cells may play a role in regulating DCs in AEDS. Malassezia yeast cells affect the NK cell-DC interaction, e.g. by decreasing NK cell cytotoxicity. The increase of CD48+, CD83+ and CD86+ DCs and induction of IL-8 production imply that NK cells activate the DCs. Furthermore, the results suggest a function of NK cell-DC interaction in shaping immune responses. Better knowledge of the activation and modulation of DCs and their function might give greater understanding of the immunological phenomenon behind the onset of AEDS. Ultimately, studies on initiation of immune responses might lead to new strategies for prevention and treatment of conditions like AEDS