Atopic dermatitis (AD) is a T cell-mediated inflammatory skin disease, with TH2 cells initiating acute flares. This inflamed skin is immediately colonized with Staphylococcus aureus, which provides potent Toll-like receptor (TLR) 2 ligands. However, the effect of TLR2 ligands on the development of TH2-mediated AD inflammation remains unclear.
The beneficial effects of nonpathogenic bacteria are increasingly being recognized. We reported in a placebo-controlled study with atopic dermatitis (AD) patients that cutaneous exposure to lysates of nonpathogenic bacteria alleviates skin inflammation. To now unravel underlying mechanisms, immune consequences of sensing nonpathogenic bacterium Vitreoscilla filiformis lysate (Vf) were characterized analyzing (1) differentiation of dendritic cells (DCs) and, consecutively, (2) effector functions of DCs and T helper (Th) cells in vitro and in a murine model of AD in NC/Nga mice in vivo. Topical treatment with Vf significantly reduced AD-like inflammation in NC/Nga mice. Importantly, cutaneous exposure to Vf in combination with the allergen FITC significantly also reduced subsequent allergen-induced dermatitis indicating active immune modulation. Indeed, innate sensing of Vf predominantly induced IL-10-producing DCs, which was dependent on Toll-like receptor 2 (TLR2) activation. Vf-induced IL-10+ DCs primed naive CD4+ T helper cells to become regulatory IFN-?(low) IL-10(high) Tr1 (type 1 regulatory T) cells. These IL-10(high) Tr1 cells were also induced by Vf in vivo and strongly suppressed T effector cells and inflammation. In conclusion, we show that innate sensing of nonpathogenic bacteria by TLR2 induces tolerogenic DCs and regulatory Tr1 cells suppressing T effector cells and cutaneous inflammation. These findings indicate a promising therapeutic strategy for inflammatory skin diseases like AD.
Psoriatic skin lesions are characterized by an inflammatory infiltrate, consisting of dendritic cells, monocytes, and both CD4(+) and CD8(+) T lymphocytes. Although the chemokines involved in the migration of CD4(+) T cells into psoriatic skin are well characterized, those regulating CD8(+) T-cell recruitment are less understood. We found that the percentages of peripheral blood CD8(+) T cells expressing CXCR6 were higher in psoriatic patients than in healthy or atopic individuals. In addition, CXCR6 expression in psoriatic patients was more abundant in the CD8(+) than in the CD4(+) T-cell compartment. CXCR6 mRNA expression was also stronger in skin CD8(+) T cells than in the corresponding blood-derived counterparts. Immunofluorescence analysis revealed profound upregulation of the CXCR6 ligand CXCL16 by monocytes, keratinocytes, and dendritic cells in psoriatic skin compared with healthy or atopic dermatitis skin. In line with this, CXCR6(+) CD8(+) T cells also were most prevalent in psoriatic skin. Furthermore, CXCL16 induced Ca(2+) influx and chemotactic migration of psoriatic skin-derived CD8(+) T cells in vitro. Most importantly, CXCL16 potently recruited human CD8(+) T cells to human skin grafts previously transplanted onto SCID mice in vivo. These investigations indicate that CXCL16-CXCR6 interactions mediate homing of CD8(+) T cells into human skin, and thereby contribute to psoriasis pathogenesis.
Casein kinase 1 alpha (CK1alpha) is a multifunctional Ser/Thr kinase that phosphorylates several substrates. Among those is beta-catenin, an important player in cell adhesion and Wnt signaling. Phosphorylation of beta-catenin by CK1alpha at Ser45 is the priming reaction for the proteasomal degradation of beta-catenin. Interestingly, aside from this role in beta-catenin degradation, very little is known about the expression and functional role of CK1alpha in tumor cells. Here, we show that CK1alpha expression in different tumor types is either strongly suppressed or completely lost during tumor progression and that CK1alpha is a key factor determining beta-catenin stability and transcriptional activity in tumor cells. CK1alpha reexpression in metastatic melanoma cells reduces growth in vitro and metastasis formation in vivo, and induces cell cycle arrest and apoptosis, whereas suppression of CK1alpha in primary melanoma cells induces invasive tumor growth. Inactivation of CK1alpha promotes tumor progression by regulating a switch in beta-catenin-mediated signaling. These results show that melanoma cells developed an efficient new mechanism to activate the beta-catenin signaling pathway and define CK1alpha as a novel tumor suppressor.
In mammalian host cells staphylococcal peptidoglycan (PGN) is recognized by Nod2. Whether PGN is also recognized by TLR2 is disputed. Here we carried out PGN co-localization and stimulation studies with TLR2 and Nod2 in wild type and mutant host cells. To exclude contamination with lipoproteins, polymeric staphylococcal PGN (PGN(pol)) was isolated from Staphylococcus aureus ?lgt (lacking lipidated prelipoproteins). PGN(pol) was biotinylated (PGN-Bio) for fluorescence monitoring with specific antibodies. Keratinocytes from murine oral epithelium (MK) readily internalized PGN-Bio in an endocytosis-like process. In wt MK, PGN(pol) induced intracellular accumulation of Nod2 and TLR2 and co-localized with Nod2 and TLR2, but not with TLR4. In TLR2-deficient MK Nod2 and in Nod2-deficient MK TLR2 was induced, indicating that PGN(pol) recognition by Nod2 is independent of TLR2 and vice versa. In both mutants IL-6 and IL-1B release was decreased by approximately 50% compared to wt MK, suggesting that the immune responses induced by Nod2 and TLR2 are comparable and that the two receptors act additively in MK. In TLR2-transfected HEK293 cells PGN(pol) induced NFkB-promoter fused luciferase expression. To support the data, co-localization and signaling studies were carried out with SHL-PGN, a lipase protein covalently tethered to PGN-fragments of varying sizes at its C-terminus. SHL-PGN also co-localized with Nod2 or TLR2 and induced their accumulation, while SHL without PGN did not. The results show that staphylococcal PGN not only co-localizes with Nod2 but also with TLR2. PGN is able to stimulate the immune system via both receptors.
Innate immune sensing of Staphylococcus aureus unravels basic mechanisms leading to either effective antibacterial immune responses or harmful inflammation. The nature and properties of S. aureus-derived pathogen-associated molecular pattern (PAMPs) are still not completely understood. We investigated the innate immune sensing of peptidoglycan (PGN) structures and subsequent immune consequences. Macromolecular PGN (PGN(polymer)) preparations activated NF-?B through human Toll-like receptors 2 (TLR2), as shown by luciferase reporter assays, and induced murine dendritic cell (DC) maturation and cytokine production. In contrast, PGN(polymer) from lgt-mutant S. aureus failed to stimulate human TLR2, demonstrating that lipoproteins within the macromolecular structures of PGN(polymer), but not PGN itself, activate TLR2. Thus, HPLC-purified monomeric PGN (PGN(monomer)) structures were investigated. Strikingly, PGN(monomer) completely lacked NF-?B activation, lacked TLR2 activity, and failed to functionally activate murine DCs. However, PGN(monomer) in concert with various TLR ligands most effectively stimulated DCs to up-regulate IL-12p70 and IL-23 by ?3- to 5-fold. Consequently, DCs coactivated by PGN(monomer) markedly up-regulated Th1 and Th17 while suppressing Th2 cell priming. Notably, PGN(monomer) failed to coactivate NOD2(-/-) DCs. This demonstrates that PGN(monomer) is a natural ligand of NOD2, which was previously only demonstrated for synthetic compounds like muramyl dipeptide. Interestingly, murine DCs lacking TLR2 remained mute in response to the combinative immune sensing of S. aureus-derived PAMPs, including PGN(monomer), providing for the first time an explanation of why S. aureus can colonize the nasal mucosa in the absence of inflammation. This is very likely based on the lack of TLR2 expression in mucosal epithelial cells under normal conditions, which determines the unresponsiveness to S. aureus PAMPs.
The PI3K pathway plays a pivotal role in the stimulation of mast cells. PI3K-dependent kinases include the serum- and glucocorticoid-inducible kinase 1 (SGK1). The present study explored the role of SGK1 in mast cell function. Mast cells were isolated from bone marrow (BMMC) of SGK1 knockout mice (sgk1(-/-)) and their wild-type littermates (sgk1(+/+)). The BMMC number as well as CD117, CD34, and FcepsilonRI expression in BMCCs were similar in both genotypes. Upon Ag stimulation of the FcepsilonRI receptor, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in sgk1(-/-) BMMCs. The currents through Ca(2+)-activated K+ channels induced by Ag were significantly higher in sgk1(+/+) BMMCs than in sgk1(-/-) BMMCs. Treatment with the Ca(2+) ionophore ionomycin (1 microM) led to activation of the K+ channels in both genotypes, indicating that the Ca(2+)-activated K+ channels are similarly expressed and sensitive to activation by Ca(2+) in sgk1(+/+) and sgk1(-/-) BMMCs, and that blunted stimulation of Ca(2+)-activated K+ channels was secondary to decreased Ca(2+) entry. Ag-IgE-induced degranulation and early IL-6 secretion were also significantly blunted in sgk1(-/-) BMMCs. The decrease in body temperature following Ag treatment, which reflects an anaphylactic reaction, was substantially reduced in sgk1(-/-) mice, pointing to impaired mast cell function in vivo. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk1(-/-) than in sgk1(+/+)mice. The observations reveal a critical role for SGK1 in ion channel regulation and the function of mast cells, and thus disclose a completely novel player in the regulation of allergic reaction.
C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.
Mast cells (MC) are key mediators of the immune system, most prominently known for their role in eliciting harmful allergic reactions. Mast cell mediator release (e.g. by degranulation) is triggered by Fc?RI recognition of antigen - IgE complexes. Until today no therapeutic targeting of this and other mast cell activation pathways is established. Among possible new candidates there are tetraspanins that have been described on MC already several years ago. Tetraspanins are transmembrane proteins acting as scaffolds, mediating local clustering of their interaction partners, and thus amplify their activities. More recently, tetraspanins were also found to exert intrinsic receptor functions. Tetraspanins have been found to be crucial components of fundamental biological processes like cell motility and adhesion. In immune cells, they not only boost the effectiveness of antigen presentation by clustering MHC molecules, they are also key players in all kinds of degranulation events and immune receptor clustering. This review focuses on the contribution of tetraspanins clustered with Fc?RI or residing in granule membranes to classical MC functions but also undertakes an outlook on the possible contribution of tetraspanins to newly described mast cell functions and discusses possible targets for drug development.
T-cell activation and the subsequent transformation of activated T cells into T-cell blasts require profound changes in cell volume. However, the impact of cell volume regulation for T-cell immunology has not been characterized. Here we studied the role of the cell-volume regulating osmolyte transporter Taut for T-cell activation in Taut-deficient mice. T-cell mediated recall responses were severely impaired in taut(-/-) mice as shown with B16 melanoma rejection and hapten-induced contact hypersensitivity. CD4(+) and CD8(+) T cells were unequivocally located within peripheral lymph nodes of unprimed taut(-/-) mice but significantly decreased in taut(-/-) compared with taut(+/+) mice following in vivo activation. Further analysis revealed that Taut is critical for rescuing T cells from activation-induced cell death in vitro and in vivo as shown with TCR, superantigen, and antigen-specific activation. Consequently, reduction of CD4(+) and CD8(+) T cells in taut(-/-) mice upon antigen challenge resulted in impaired in vivo generation of T-cell memory. These findings disclose for the first time that volume regulation in T cells is an element in the regulation of adaptive immune responses and that the osmolyte transporter Taut is crucial for T-cell survival and T-cell mediated immune reactions.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.