Human IL-31 is induced by IL-4 and promotes TH2-driven inflammation.

BACKGROUND: The pruritic cytokine IL-31 has been shown to be expressed by murine activated effector T Lymphocytes of a TH2 phenotype. Like IL-17 and IL-22, IL-31 is a tissue-signaling cytokine the receptor of which is mainly found on nonimmune cells. An overabundance of IL-31 has been shown in patients with atopic disorders, including dermatitis, as well as asthma, and therefore represents a promising drug target, although its regulation in the context of the human TH2 clusters is not yet known. OBJECTIVE: We sought to address the gene regulation of human IL-31 and to test whether IL-31 possesses a similar proallergic function as members of the human TH2 cytokine family, such as IL-4, IL-5, and IL-13. METHODS: Polyclonal and purified protein derivative of tuburculin-specific T-cell clones were generated. TH phenotype was determined, and IL-31 was measured by means of ELISA. Gene expression of primary bronchial epithelial cells treated with IL-31 was also measured. RESULTS: IL-31 was expressed by all of the TH2 clones and not by TH1, TH17, or TH22. This expression was dependent on autocrine IL-4 expression from these clones because it could be reduced if blocking antibodies to IL-4 were present. Interestingly, TH1 clones were able to express IL-31 if IL-4 was added to culture. This IL-31 expression was transient and did not affect the phenotype of the TH1 clones. IL-31 was able to induce proinflammatory genes, such as CCL2 and granulocyte colony-stimulating factor. CONCLUSION: IL-31 is not a TH2 cytokine in the classical sense but is likely to be expressed by a number of cells in an allergic situation in which IL-4 is present and possibly contribute to the allergic reaction.

IL-22 suppresses IFN-γ-mediated lung inflammation in asthmatic patients.

BACKGROUND: IL-22 controls tissue homeostasis by both proinflammatory and anti-inflammatory effects. However, the anti-inflammatory mechanisms of IL-22 remain poorly investigated. OBJECTIVE: We sought to investigate the anti-inflammatory role for IL-22 in human asthma. METHODS: T-cell lines derived from lung biopsy specimens of asthmatic patients were characterized by means of flow cytometry. Human bronchial epithelial cells from healthy and asthmatic subjects were stimulated with IL-22, IFN-γ, or the combination of both cytokines. Effects of cytokine stimulation were investigated by using whole-genome analysis, ELISA, and flow cytometry. The functional consequence of cytokine stimulation was evaluated in an in vitro wound repair model and T cell-mediated cytotoxicity experiments. In vivo cytokine expression was measured by using immunohistochemistry and Luminex assays in bronchoalveolar lavage fluid of healthy and asthmatic patients. RESULTS: The current study identifies a tissue-restricted antagonistic interplay of IL-22 and the proinflammatory cytokine IFN-γ. On the one hand, IFN-γ antagonized IL-22-mediated induction of the antimicrobial peptide S100A7 and epithelial cell migration in bronchial epithelial cells. On the other hand, IL-22 decreased epithelial susceptibility to T cell-mediated cytotoxicity by inhibiting the IFN-γ-induced expression of MHC-I, MHC-II, and CD54/intercellular adhesion molecule 1 molecules. Likewise, IL-22 inhibited IFN-γ-induced secretion of the proinflammatory chemokines CCL5/RANTES and CXCL10/interferon-inducible protein 10 in vitro. Consistently, the IL-22 expression in bronchoalveolar lavage fluid of asthmatic patients inversely correlated with the expression of CCL5/RANTES and CXCL10/interferon-inducible protein 10 in vivo. CONCLUSIONS: IL-22 might control the extent of IFN-γ-mediated lung inflammation and therefore play a tissue-restricted regulatory role.

IL-22 and TNF-α represent a key cytokine combination for epidermal integrity during infection with Candida albicans.

T cells exercise their full impact on target cells through a combination of secreted cytokines. The recently described T helper cell subset Th22 is characterized by a combinatorial secretion of IL-22 and TNF-α. Here, we demonstrate that IL-22 increases the TNF-α-dependent induction and secretion of several immune-modulatory molecules such as initial complement factors C1r and C1s, antimicrobial peptides S100A7 and HBD-2 (human ß defensin 2), and antimicrobial chemokines CXCL-9/-10/-11 in primary human keratinocytes. The synergism of IL-22 and TNF-α is transmitted intracellularly by MAP kinases and downstream by transcription factors of the AP-1 family. The induction of innate immunity is relevant in an in vitro infection model, where keratinocytes stimulated with Th22 supernatants or recombinant IL-22 plus TNF-α effectively inhibit the growth of Candida albicans and maintain survival of epithelia. Accordingly, the combinatorial stimulation of keratinocytes with IL-22 and TNF-α most efficiently conserves the integrity of the epidermal barrier in a three-dimensional skin infection model as compared with IFN-γ, IL-17, IL-22 or TNF-α alone. In summary, we demonstrate that IL-22 and TNF-α represent a potent, synergistic cytokine combination for cutaneous immunity.

CD56highCD16-CD62L- NK cells accumulate in allergic contact dermatitis and contribute to the expression of allergic responses.

Allergic contact dermatitis is a common disease caused by an exaggerated T cell-mediated immune response to skin-applied haptens. We show in this study that NK cells affect skin immune responses to haptens by releasing type 1 cytokines and inducing keratinocytes apoptosis. Immunohistochemical stainings demonstrated that NK lymphocytes constitute approximately 10% of the inflammatory infiltrate mostly distributed in the superficial dermis and in the epidermis at the site of intense spongiotic changes. More than 90% of NK cells isolated from allergic contact dermatitis skin showed a CD3-CD56(high)CD16- phenotype by FACS analysis. In addition, they uniformly expressed NKG2A, intermediate to high levels of perforin, and the activating receptors, NKG2D, NKp44, and NKp46, but lacked NKp30 and killer Ig-related receptors. Skin NK lymphocytes displayed a CXCR3+CCR6+CCR5+ chemokine receptor asset for homing into inflamed skin, but not CD62L and CCR7 for lymph node homing. When NK cells from nickel-allergic donors were exposed in vitro to the metal, they failed to proliferate, to upregulate CD69, and to release IFN-gamma, thus indicating that NK lymphocytes do not exhibit memory-like properties to haptens. However, IL-2 released by hapten-driven T lymphocytes rapidly induced the release of IFN-gamma by NK cells and promoted the NK-mediated apoptosis of autologous keratinocytes in a hapten-independent manner. Our findings underline the importance of the interaction between innate and adaptive immune mechanisms for amplification of skin allergic responses to haptens and full expression of allergic contact dermatitis.

IL-17 amplifies human contact hypersensitivity by licensing hapten nonspecific Th1 cells to kill autologous keratinocytes.

Th17 is a newly identified lineage of effector T cells involved in autoimmunity and immune responses to pathogens. We demonstrate in this study the pathogenic role of IL-17-producing CD4(+) T lymphocytes in allergic contact dermatitis (ACD) to skin-applied chemicals. IL-17(+) T cells infiltrate ACD reactions and predominantly distribute at the site of heavy spongiosis. Skin IL-17(+) T cells were functionally and phenotypically heterogeneous: although pure Th17 prevailed in ACD skin, hapten responsiveness was restricted to Th1/IL-17 (IFN-gamma(+)IL-17(+)) and Th0/IL-17 (IFN-gamma(+)IL-17(+)IL-4(+)) fractions, and to lesser extent Th2/IL-17 cells. In the IFN-gamma-dominated ACD environment, IL-17-releasing T cells affect immune function of keratinocytes by promoting CXCL8, IL-6, and HBD-2 production. In addition, compared with Th1, supernatants from Th1/IL-17 T cells were much more efficient in inducing ICAM-1 expression on keratinocytes and keratinocyte-T cell adhesiveness in vitro. As a consequence, exposure to combined IFN-gamma and IL-17 rendered keratinocytes susceptible to ICAM-1-dependent Ag nonspecific T cell killing. Thus, IL-17 efficiently amplifies the allergic reaction by rendering virtually all of the T lymphocytes recruited at the site of skin inflammation capable to directly contribute to tissue damage.

CD56 highCD16 - NK cell involvement in cutaneous lichen planus.

Lichen planus is an inflammatory disease of the skin and mucous membranes characterized by vacuolization of basal keratinocytes associated with a prominent junctional lymphocyte infiltrate which comprises T lymphocytes, NK cells, myeloid and plasmacytoid dendritic cells. Basal keratinocyte damage is considered as being a consequence of a lymphocytic cytotoxic attack, mostly mediated by perforin+CD8+ T lymphocytes. NK cells have been described to infiltrate inflamed skin and significantly contribute to the amplification of immune-mediated skin diseases, thanks to their cytotoxic activity and the release of pro-inflammatory cytokines. Here, we investigated the characteristics and functional properties of NK lymphocytes involved in lichen planus. Double staining immunohistochemistry showed a considerable number (6.42 ± 2.2% of the total cellular infiltrate) of CD3-CD56+ cells in early lichen planus lesions, mostly distributed in the papillary dermis and at the epidermal-dermal interface. Skin NK cells isolated from lichen planus lesions belong to the CD56highCD16- subset, are highly positive for perforin and natural cytotoxic receptors NKG2D and NKp44, and, in accordance with their phenotype, are negative for KIRs receptors CD158a and CD158b. Skin CD56highCD16- NK cells display a CCR6+CXCR3+CCR5+ChemR23+ chemokine receptor asset for homing into inflamed skin. In terms of cytokine release, skin CD56highCD16- NK cells are able to secrete IFN-γ, TNF-α and hardly release IL-22, IL-17 and IL-4. Overall, our data propose a pro-inflammatory role of NK lymphocytes in lichen planus.

IL-17 in atopic eczema: linking allergen-specific adaptive and microbial-triggered innate immune response.

BACKGROUND: Patients with atopic eczema (AE) regularly experience colonization with Staphylococcus aureus that is directly correlated with the severity of eczema. Recent studies show that an impaired IL-17 immune response results in diseases associated with chronic skin infections. OBJECTIVE: We sought to elucidate the effect of IL-17 on antimicrobial immune responses in AE skin. METHODS: T cells infiltrating atopy patch test (APT) reactions were characterized for IL-17 secretion to varying stimuli. IL-17-dependent induction of the antimicrobial peptide human beta-defensin 2 (HBD-2) in keratinocytes was investigated. RESULTS: Approximately 10% of APT-infiltrating T cells secreted IL-17 after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation. Among these, 33% belonged to the newly characterized subtype T(H)2/IL-17. Despite the capacity to secrete IL-17, specific T-cell clones released only low amounts of IL-17 on cognate allergen stimulation, whereas IL-4, IFN-gamma, or both were efficiently induced. IL-17 secretion was not enhanced by IL-23, IL-1 beta, or IL-6 but was enhanced by the S aureus-derived superantigen staphylococcal enterotoxin B. Both healthy and AE keratinocytes upregulated HBD-2 in response to IL-17, but coexpressed IL-4/IL-13 partially inhibited this effect. In vivo, additional application of staphylococcal enterotoxin B induced IL-17 in APT reactions, whereas IL-4, IFN-gamma, and IL-10 were marginally regulated. Induced IL-17 upregulated HBD-2 in human keratinocytes in vivo. CONCLUSION: IL-17-capable T cells, in particular T(H)2/IL-17 cells, infiltrate acute AE reactions. Although IL-17 secretion by specific T cells is tightly regulated, it can be triggered by bacteria-derived superantigens. The ineffective IL-17-dependent upregulation of HBD-2 in patients with AE is due to a partial inhibition by the type 2 microenvironment, which could partially explain why patients with AE do not clear S aureus.

Microbe-host interplay in atopic dermatitis and psoriasis.

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.

Optimizing of the basophil activation test: Comparison of different basophil identification markers.

BACKGROUND: Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement. METHODS: Ten patients with a grass pollen allergy and three controls were included in the study. Basophilic cells were gated by using anti-CCR3, anti-IgE, anti-CRTH2, anti-CD203c, and anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the allergen extract Phleum pratense. The activation marker anti-CD63 was used. RESULTS: The highest relative number of basophils was found with anti-CCR3+ cells, anti-IgE+ and anti-IgE+ /anti-CD203c+ cells, the lowest with CRTH2+/CD203c+/CD3- cells. A very good and good concordance of CCR3+ cells was seen with CCR3+/CD3- cells and CRTH2+/CD203c+/CD3- cells in all experiments. The contamination of the CCR3+ population with CD3+ cells and the contamination of the IgE+-population with CCR3- cells and CD203- cells were the lowest compared to all other marker combinations. CONCLUSIONS: As the highest relative number of basophils was identified by anti-CCR3 followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen.

Optimizing of the basophil activation test: Comparison of different basophil identification markers.

Background Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement. Methods Ten patients with a grass pollen allergy and 3 controls were included in the study. Basophilic cells were gated by using anti-CCR3647, anti-IgE, anti-CRTH2, anti-CD203c und anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and the allergen extract Phleum pratense. The activation marker anti-CD63 was used. Results The highest relative number of basophils was found with anti-CCR3+ cells, anti-IgE+ and anti-IgE+ /anti-CD203c+ cells, the lowest with CRTH2+ /CD203c+ /CD3- cells. A very good and good concordance of CCR3+ cells was seen with CCR3+ /CD3- cells and CRTH2+ /CD203c+ /CD3- cells in all experiments. The contamination of the CCR3+ population with CD3+ cells and the contamination of the IgE+ -population with CCR3- cells and CD203- cells were the lowest compared to all other marker combinations. Conclusions As the highest relative number of basophils was identified by anti-CD193 (CCR3) followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen. © 2014 Clinical Cytometry Society.

Basophil activation test using recombinant allergens: highly specific diagnostic method complementing routine tests in wasp venom allergy.

BACKGROUND: Skin testing can expose allergic subjects to potential systemic reactions, sensitization against unrelated proteins, and increased risk of future sting reactions. Therefore the continuous improvement of in vitro diagnostic methods is desirable. Recombinant allergens have been shown to improve the sensitivity of specific IgE (sIgE) detection in vitro whilst no data is available regarding their application and reliability in basophil activation test (BAT). Here we aimed to compare the specificity and sensitivity of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 in both specific IgE (sIgE) detection in vitro and basophil activation test. METHODS: sIgE detection by ELISA or ImmunoCAP and BAT towards the panel of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 were performed in 43 wasp venom allergic patients with a history of anaphylactic reaction and sIgE seropositivity, as well as 17 controls defined as subjects with a history of repetitive wasp stings but absence of any allergic symptom. RESULTS: The BAT performed with the recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to the respective sIgE detection in serum. CONCLUSIONS: BAT performed with the recombinant allergens Ves v 5, Ves v 3 and Ves v 1 provides an emerging highly specific in vitro method for the detection of wasp venom allergy, compared to the sIgE detection. Recombinant allergens applied to BAT represent a step forward in developing reliable in vitro tests for specific diagnosis of allergy.

Intraindividual genome expression analysis reveals a specific molecular signature of psoriasis and eczema.

Previous attempts to gain insight into the pathogenesis of psoriasis and eczema by comparing their molecular signatures were hampered by the high interindividual variability of those complex diseases. In patients affected by both psoriasis and nonatopic or atopic eczema simultaneously (n = 24), an intraindividual comparison of the molecular signatures of psoriasis and eczema identified genes and signaling pathways regulated in common and exclusive for each disease across all patients. Psoriasis-specific genes were important regulators of glucose and lipid metabolism, epidermal differentiation, as well as immune mediators of T helper 17 (TH17) responses, interleukin-10 (IL-10) family cytokines, and IL-36. Genes in eczema related to epidermal barrier, reduced innate immunity, increased IL-6, and a TH2 signature. Within eczema subtypes, a mutually exclusive regulation of epidermal differentiation genes was observed. Furthermore, only contact eczema was driven by inflammasome activation, apoptosis, and cellular adhesion. On the basis of this comprehensive picture of the pathogenesis of psoriasis and eczema, a disease classifier consisting of NOS2 and CCL27 was created. In an independent cohort of eczema (n = 28) and psoriasis patients (n = 25), respectively, this classifier diagnosed all patients correctly and also identified initially misdiagnosed or clinically undifferentiated patients.

CX3CL1 (fractalkine) and its receptor CX3CR1 regulate atopic dermatitis by controlling effector T cell retention in inflamed skin.

Atopic dermatitis (AD) is a chronic allergic dermatosis characterized by epidermal thickening and dermal inflammatory infiltrates with a dominant Th2 profile during the acute phase, whereas a Th1 profile is characteristic of the chronic stage. Among chemokines and chemokine receptors associated with inflammation, increased levels of CX3CL1 (fractalkine) and its unique receptor, CX3CR1, have been observed in human AD. We have thus investigated their role and mechanism of action in experimental models of AD and psoriasis. AD pathology and immune responses, but not psoriasis, were profoundly decreased in CX3CR1-deficient mice and upon blocking CX3CL1-CX3CR1 interactions in wild-type mice. CX3CR1 deficiency affected neither antigen presentation nor T cell proliferation in vivo upon skin sensitization, but CX3CR1 expression by both Th2 and Th1 cells was required to induce AD. Surprisingly, unlike in allergic asthma, where CX3CL1 and CX3CR1 regulate the pathology by controlling effector CD4(+) T cell survival within inflamed tissues, adoptive transfer experiments established CX3CR1 as a key regulator of CD4(+) T cell retention in inflamed skin, indicating a new function for this chemokine receptor. Therefore, although CX3CR1 and CX3CL1 act through distinct mechanisms in different pathologies, our results further indicate their interest as promising therapeutic targets in allergic diseases.

Allergic contact dermatitis in psoriasis patients: typical, delayed, and non-interacting.

Psoriasis is characterized by an apoptosis-resistant and metabolic active epidermis, while a hallmark for allergic contact dermatitis (ACD) is T cell-induced keratinocyte apoptosis. Here, we induced ACD reactions in psoriasis patients sensitized to nickel (n = 14) to investigate underlying mechanisms of psoriasis and ACD simultaneously. All patients developed a clinically and histologically typical dermatitis upon nickel challenge even in close proximity to pre-existing psoriasis plaques. However, the ACD reaction was delayed as compared to non-psoriatic patients, with a maximum intensity after 7 days. Whole genome expression analysis revealed alterations in numerous pathways related to metabolism and proliferation in non-involved skin of psoriasis patients as compared to non-psoriatic individuals, indicating that even in clinically non-involved skin of psoriasis patients molecular events opposing contact dermatitis may occur. Immunohistochemical comparison of ACD reactions as well as in vitro secretion analysis of lesional T cells showed a higher Th17 and neutrophilic migration as well as epidermal proliferation in psoriasis, while ACD reactions were dominated by cytotoxic CD8+ T cells and a Th2 signature. Based on these findings, we hypothesized an ACD reaction directly on top of a pre-existing psoriasis plaque might influence the clinical course of psoriasis. We observed a strong clinical inflammation with a mixed psoriasis and eczema phenotype in histology. Surprisingly, the initial psoriasis plaque was unaltered after self-limitation of the ACD reaction. We conclude that sensitized psoriasis patients develop a typical, but delayed ACD reaction which might be relevant for patch test evaluation in clinical practice. Psoriasis and ACD are driven by distinct and independent immune mechanisms.

Human T cell priming assay (hTCPA) for the identification of contact allergens based on naive T cells and DC--IFN-γ and TNF-α readout.

Many small protein reactive organic and inorganic chemicals can cause allergic contact dermatitis, a T cell mediated inflammatory skin disease. In vitro alternatives to animal testing are needed for the identification of chemicals that pose such risks to human health. We here publish the standard operation procedure for a human T cell priming assay developed primarily for the identification of contact allergens within the integrated EU project Sens-it-iv. This multiparametric flow cytometry based assay identifies chemical specific T cells based on their frequency and antigen-specific production of the cytokines IFN-γ and TNF-α at the single cell level. Using sorted naïve T cells and monocyte-derived dendritic cells pulsed with the test chemical or with chemical-protein conjugates, the successful priming of an antigen-specific T cell response is controlled after antigen-specific restimulation by cytokine readout. As the most specific response of the immune system to contact allergens the analysis of the chemical-specific T cell response may be a useful in vitro assay for hazard identification in immunotoxicology. This assay may be a valuable, highly specific element of an integrated testing strategy for the identification of chemicals and drugs that cause T cell mediated respiratory or gastrointestinal tract hypersensitivities or adverse drug reactions.

Th17 and Th22 in skin allergy.

Development of eczematous skin reactions depends on disease-specific and time-dependent recruitment of a variety of leukocytes affecting resident skin cells through cytotoxic mechanisms and release of cytokines. Th17 and Th22, defined as RORC+IL-17+ and IL-17-IFN-γ-IL-22+ cells, respectively, belong to a newly identified class of lymphocytes specifically involved in dialogue with non-immune cells. In line with this function, both Th17 and Th22 cells are enriched in many immune-mediated skin diseases, such as a topic dermatitis, allergic contact dermatitis and psoriasis. Both IL-17 and IL-22 activate keratinocyte innate immune defenses, thus protecting the skin from pathogen invasion. However, Th17 and Th22 differ in their proinflammatory functions, being prominent in the first T cell subset and occasional/opportunistic in the second T cell subset. Most of the proinflammatory functions of Th17 depend on the synergic activity of IFN-γ and IL-17 on target cells. Together with IFN-γ, IL-17 strongly enhances adhesion molecules on keratinocytes, thus promoting T cell-keratinocyte adhesion and T cell-mediated cytotoxicity, resulting in keratinocyte apoptosis. In contrast, Th22 cells guarantee skin integrity by inducing keratinocyte proliferation and migration. However, in inflamed skin, Th22 could contribute to the amplification of immune responses by enhancing the TNF-α-induced cytokines and chemokines released by keratinocytes.