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.

Hematopoietic ChemR23 (Chemerin Receptor 23) Fuels Atherosclerosis by Sustaining an M1 Macrophage-Phenotype and Guidance of Plasmacytoid Dendritic Cells to Murine Lesions-Brief Report.

Objective- Expression of the chemokine-like receptor ChemR23 (chemerin receptor 23) has been specifically attributed to plasmacytoid dendritic cells (pDCs) and macrophages and ChemR23 has been suggested to mediate an inflammatory immune response in these cells. Because chemokine receptors are important in perpetuating chronic inflammation, we aimed to establish the role of ChemR23-deficiency on macrophages and pDCs in atherosclerosis. Approach and Results- ChemR23-knockout/knockin mice expressing eGFP (enhanced green fluorescent protein) were generated and after crossing with apolipoprotein E-deficient ( Apoe-/- ChemR23 e/e) animals were fed a western-type diet for 4 and 12 weeks. Apoe-/- ChemR23 e/e mice displayed reduced lesion formation and reduced leukocyte adhesion to the vessel wall after 4 weeks, as well as diminished plaque growth, a decreased number of lesional macrophages with an increased proportion of M2 cells and a less inflammatory lesion composition after 12 weeks of western-type diet feeding. Hematopoietic ChemR23-deficiency similarly reduced atherosclerosis. Additional experiments revealed that ChemR23-deficiency induces an alternatively activated macrophage phenotype, an increased cholesterol efflux and a systemic reduction in pDC frequencies. Consequently, expression of the pDC marker SiglecH in atherosclerotic plaques of Apoe-/- ChemR23 e/e mice was declined. ChemR23-knockout pDCs also exhibited a reduced migratory capacity and decreased CCR (CC-type chemokine receptor)7 expression. Finally, adoptive transfer of sorted wild-type and knockout pDCs into Apoe-/- recipient mice revealed reduced accumulation of ChemR23-deficient pDCs in atherosclerotic lesions. Conclusions- Hematopoietic ChemR23-deficiency increases the proportion of alternatively activated M2 macrophages in atherosclerotic lesions and attenuates pDC homing to lymphatic organs and recruitment to atherosclerotic lesions, which synergistically restricts atherosclerotic plaque formation and progression.

New mechanism underlying IL-31-induced atopic dermatitis.

BACKGROUND: TH2 cell-released IL-31 is a critical mediator in patients with atopic dermatitis (AD), a prevalent and debilitating chronic skin disorder. Brain-derived natriuretic peptide (BNP) has been described as a central itch mediator. The importance of BNP in peripheral (skin-derived) itch and its functional link to IL-31 within the neuroimmune axis of the skin is unknown. OBJECTIVE: We sought to investigate the function of BNP in the peripheral sensory system and skin in IL-31-induced itch and neuroepidermal communication in patients with AD. METHODS: Ca2+ imaging, immunohistochemistry, quantitative real-time PCR, RNA sequencing, knockdown, cytokine/phosphokinase arrays, enzyme immune assay, and pharmacologic inhibition were performed to examine the cellular basis of the IL-31-stimulated, BNP-related itch signaling in dorsal root ganglionic neurons (DRGs) and skin cells, transgenic AD-like mouse models, and human skin of patients with AD and healthy subjects. RESULTS: In human DRGs we confirmed expression and co-occurrence of oncostatin M receptor ß subunit and IL-31 receptor A in a small subset of the neuronal population. Furthermore, IL-31 activated approximately 50% of endothelin-1-responsive neurons, and half of the latter also responded to histamine. In murine DRGs IL-31 upregulated Nppb and induced soluble N-ethylmaleimide-sensitive factor activating protein receptor-dependent BNP release. In Grhl3PAR2/+ mice house dust mite-induced severe AD-like dermatitis was associated with Nppb upregulation. Lesional IL-31 transgenic mice also exhibited increased Nppb transcripts in DRGs and the skin; accordingly, skin BNP receptor levels were increased. Importantly, expression of BNP and its receptor were increased in the skin of patients with AD. In human skin cells BNP stimulated a proinflammatory and itch-promoting phenotype. CONCLUSION: For the first time, our findings show that BNP is implicated in AD and that IL-31 regulates BNP in both DRGs and the skin. IL-31 enhances BNP release and synthesis and orchestrates cytokine and chemokine release from skin cells, thereby coordinating the signaling pathways involved in itch. Inhibiting peripheral BNP function might be a novel therapeutic strategy for AD and pruritic conditions.

The pruritus- and TH2-associated cytokine IL-31 promotes growth of sensory nerves.

BACKGROUND: Pruritus is a cardinal symptom of atopic dermatitis, and an increased cutaneous sensory network is thought to contribute to pruritus. Although the immune cell-IL-31-neuron axis has been implicated in severe pruritus during atopic skin inflammation, IL-31's neuropoietic potential remains elusive. OBJECTIVE: We sought to analyze the IL-31-related transcriptome in sensory neurons and to investigate whether IL-31 promotes sensory nerve fiber outgrowth. METHODS: In vitro primary sensory neuron culture systems were subjected to whole-transcriptome sequencing, ingenuity pathway analysis, immunofluorescence, and nerve elongation, as well as branching assays after IL-31 stimulation. In vivo we investigated the cutaneous sensory neuronal network in wild-type, Il31-transgenic, and IL-31 pump-equipped mice. RESULTS: Transgenic Il31 overexpression and subcutaneously delivered IL-31 induced an increase in the cutaneous nerve fiber density in lesional skin in vivo. Transcriptional profiling of IL-31-activated dorsal root ganglia neurons revealed enrichment for genes promoting nervous system development and neuronal outgrowth and negatively regulating cell death. Moreover, the growth cones of primary small-diameter dorsal root ganglia neurons showed abundant IL-31 receptor α expression. Indeed, IL-31 selectively promoted nerve fiber extension only in small-diameter neurons. Signal transducer and activator of transcription 3 phosphorylation mediated IL-31-induced neuronal outgrowth, and pharmacologic inhibition of signal transducer and activator of transcription 3 completely abolished this effect. In contrast, transient receptor potential cation channel vanilloid subtype 1 channels were dispensable for IL-31-induced neuronal sprouting. CONCLUSIONS: The pruritus- and TH2-associated novel cytokine IL-31 induces a distinct transcriptional program in sensory neurons, leading to nerve elongation and branching both in vitro and in vivo. This finding might help us understand the clinical observation that patients with atopic dermatitis experience increased sensitivity to minimal stimuli inducing sustained itch.

A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: Involvement of TRPV1 and TRPA1.

BACKGROUND: Although the cytokine IL-31 has been implicated in inflammatory and lymphoma-associated itch, the cellular basis for its pruritic action is yet unclear. OBJECTIVE: We sought to determine whether immune cell-derived IL-31 directly stimulates sensory neurons and to identify the molecular basis of IL-31-induced itch. METHODS: We used immunohistochemistry and quantitative real-time PCR to determine IL-31 expression levels in mice and human subjects. Immunohistochemistry, immunofluorescence, quantitative real-time PCR, in vivo pharmacology, Western blotting, single-cell calcium imaging, and electrophysiology were used to examine the distribution, functionality, and cellular basis of the neuronal IL-31 receptor α in mice and human subjects. RESULTS: Among all immune and resident skin cells examined, IL-31 was predominantly produced by TH2 and, to a significantly lesser extent, mature dendritic cells. Cutaneous and intrathecal injections of IL-31 evoked intense itch, and its concentrations increased significantly in murine atopy-like dermatitis skin. Both human and mouse dorsal root ganglia neurons express IL-31RA, largely in neurons that coexpress transient receptor potential cation channel vanilloid subtype 1 (TRPV1). IL-31-induced itch was significantly reduced in TRPV1-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)-deficient mice but not in c-kit or proteinase-activated receptor 2 mice. In cultured primary sensory neurons IL-31 triggered Ca(2+) release and extracellular signal-regulated kinase 1/2 phosphorylation, inhibition of which blocked IL-31 signaling in vitro and reduced IL-31-induced scratching in vivo. CONCLUSION: IL-31RA is a functional receptor expressed by a small subpopulation of IL-31RA(+)/TRPV1(+)/TRPA1(+) neurons and is a critical neuroimmune link between TH2 cells and sensory nerves for the generation of T cell-mediated itch. Thus targeting neuronal IL-31RA might be effective in the management of TH2-mediated itch, including atopic dermatitis and cutaneous T-cell lymphoma.

Proteinase-activated receptor-2 agonist activates anti-influenza mechanisms and modulates IFNγ-induced antiviral pathways in human neutrophils.

Proteinase-activated receptor-2 (PAR2) is expressed by human leukocytes and participates in the development of inflammatory diseases. Recent studies demonstrated an ability of PAR2 agonist to enhance IFNγ-induced antiviral responses of human leukocytes. However, the precise cellular antiviral defense mechanisms triggered in leukocytes after stimulation with IFNγ and/or PAR2 agonist remain elusive. Therefore, we aimed to identify neutrophil defense mechanisms involved in antiviral resistance. Here we demonstrated that PAR2 agonist enhanced IFNγ-related reduction of influenza A virus (IAV) replication in human neutrophils. PAR2-mediated decrease in IAV replication was associated with reduced NS-1 transcription. Moreover, PAR2-dependent neutrophil activation resulted in enhanced myeloperoxidase degranulation and extracellular myeloperoxidase disrupted IAV. The production of ROS was elevated in response to PAR2 activation. Interestingly, IFNγ did not influence both effects: PAR2 agonist-triggered myeloperoxidase (MPO) release and reactive oxygen species (ROS) production, which are known to limit IAV infections. In contrast, orthomyxovirus resistance gene A (MxA) protein expression was synergistically elevated through PAR2 agonist and IFNγ in neutrophils. Altogether, these findings emphasize two PAR2-controlled antiviral mechanisms that are independent of or modulated by IFNγ.

New tools for assessing the individual risk of metastasis in renal cell carcinoma.

Localized renal cell carcinoma (RCC) progresses to metastatic disease in 20-40 % after surgical resection. Affected patients might benefit from adjuvant treatment and have to be reliably identified for treatment indication. However, existing molecular markers and classification nomograms lack sufficient validity for clinical application so far. Therefore, in order to improve diagnostic tools for the identification of patients at risk, we tested invasiveness and the capability to activate vascular endothelium of primary RCC cells as tumor specific functional parameters. As a parameter for cell invasiveness the ability of RCC cells to break-down transepithelial electrical resistance (TEER) of an epithelial cell monolayer was tested. Loss of resistance, calculated as invasivity index, resembled the degree of cell invasiveness. In addition, secretion of Von Willebrand Factor by endothelial cells incubated with RCC cell supernatant was measured as a surrogate marker for endothelial cell activation. TEER-assay results matched clinical status of disease in 9 out of 12 cases. Metastatic tumors and less differentiated tumors had a significant increase of invasivity index (p = 0.007; p = 0.034). Endothelial cell activation and clinical outcome matched in 5 out of 9 samples. In addition, tumor cell induced endothelial cell activation significantly correlated to the pathologic T classification status of RCC tumors (p = 0.009). Taken together, our study validated endothelial cell activation analysis and cell invasiveness as solitary prognostic markers for tumor dissemination. TEER-analysis has proven to be a useful functional assay giving highly relevant individual information on functional tumor cell characteristics that add to pathologic evaluation.

α-1-Antitrypsin and IFN-γ reduce the severity of IC-mediated vasculitis by regulation of leukocyte recruitment in vivo.

IC-mediated vasculitis (ICV) can be life threatening. The cellular and immune mechanisms controlling ICV are poorly understood. Therefore, we investigated the role of α-1-antitrypsin (α1AT) and IFN-γ in reducing the severity of ICV in a mouse model in vivo. To induce ICV, mice were challenged with the reverse passive Arthus reaction (RPA), the prototypic in vivo model for leukocytoclastic vasculitis (LcV), and the modulation of vascular permeability, edema formation, and leukocyte recruitment was studied. To further analyze the dynamics of RPA, we applied intravital microscopy in the dorsal skinfold chamber. α1AT continuously led to reduced leukocyte recruitment. α1AT interfered with neutrophil recruitment through a KC-dependent mechanism and reduced KC-elicited neutrophil activation. In contrast to α1AT, IFN-γ-reduced leukocyte recruitment during RPA was clearly independent of KC. We also revealed that the recruitment of neutrophils during RPA was a prerequisite for full KC expression. Thus, therapeutic administration of α1AT and IFN-γ might be beneficial for limiting the duration and severity of ICV.

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor.

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with ß-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of ß-arrestin1 and PP2A with noninternalized NK(1)R. ß-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that ß-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping ß-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires ß-arrestin1. ECE-1 promotes this process by releasing ß-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

Role of proteinase-activated receptor-2 in anti-bacterial and immunomodulatory effects of interferon-γ on human neutrophils and monocytes.

Recent studies show that proteinase-activated receptor-2 (PAR(2)) contributes to the development of inflammatory responses. However, investigations into the precise role of PAR(2) activation in the anti-microbial defence of human leucocytes are just beginning. We therefore evaluated the contribution of PAR(2) to the anti-microbial response of isolated human innate immune cells. We found that PAR(2) agonist, acting alone, enhances phagocytosis of Staphylococcus aureus and killing of Escherichia coli by human leucocytes, and that the magnitude of the effect is similar to that of interferon-γ (IFN-γ). However, co-application of PAR(2) -cAP and IFN-γ did not enhance the phagocytic and bacteria-killing activity of leucocytes beyond that triggered by either agonist alone. On the other hand, IFN-γ enhances PAR(2) agonist-induced monocyte chemoattractant protein 1 (MCP-1) secretion by human neutrophils and monocytes. Furthermore, phosphoinositide-3 kinase and janus kinase molecules are involved in the synergistic effect of PAR(2) agonist and IFN-γ on MCP-1 secretion. Our findings suggest a potentially protective role of PAR(2) agonists in the anti-microbial defence established by human monocytes and neutrophils.

Interferon-γ induces upregulation and activation of the interleukin-31 receptor in human dermal microvascular endothelial cells.

Interleukin-31 (IL-31), a recently discovered cytokine derived from T helper cells, is involved in chronic dermatitis and pruritus. This study demonstrates for the first time that the IL-31 receptor complex for IL-31 is substantially upregulated in human dermal microvascular endothelial cells after stimulation with interferon-γ (IFN-γ). Activation of the IL-31 receptor complex results in the induction of the intracellular ERK1/2 signaling pathway and downregulation of IFN-γ-induced monokine induced by IFN-γ expression. Inhibitor studies revealed that the IFN-γ-induced IL-31RA upregulation is processed via JNK and PI3 kinase activation. In sum, our study points toward an interaction between the T(H) 1-derived cytokine IFN-γ and the T(H) 2-derived cytokine IL-31 on endothelial cells.

Intracellular degradation of somatostatin-14 following somatostatin-receptor3-mediated endocytosis in rat insulinoma cells.

Somatostatin receptor (SSTR) endocytosis influences cellular responsiveness to agonist stimulation and somatostatin receptor scintigraphy, a common diagnostic imaging technique. Recently, we have shown that SSTR1 is differentially regulated in the endocytic and recycling pathway of pancreatic cells after agonist stimulation. Additionally, SSTR1 accumulates and releases internalized somatostatin-14 (SST-14) as an intact and biologically active ligand. We also demonstrated that SSTR2A was sequestered into early endosomes, whereas internalized SST-14 was degraded by endosomal peptidases and not routed into lysosomal degradation. Here, we examined the fate of peptide agonists in rat insulinoma cells expressing SSTR3 by biochemical methods and confocal laser scanning microscopy. We found that [(125)I]Tyr11-SST-14 rapidly accumulated in intracellular vesicles, where it was degraded in an ammonium chloride-sensitive manner. In contrast, [(125)I]Tyr1-octreotide accumulated and was released as an intact peptide. Rhodamine-B-labeled SST-14, however, was rapidly internalized into endosome-like vesicles, and fluorescence signals colocalized with the lysosomal marker protein cathepsinD. Our data show that SST-14 was cointernalized with SSTR3, was uncoupled from the receptor, and was sorted into an endocytic degradation pathway, whereas octreotide was recycled as an intact peptide. Chronic stimulation of SSTR3 also induced time-dependent downregulation of the receptor. Thus, the intracellular processing of internalized SST-14 and the regulation of SSTR3 markedly differ from the events mediated by the other SSTR subtypes.

Agonists of proteinase-activated receptor-2 enhance IFN-gamma-inducible effects on human monocytes: role in influenza A infection.

Proteinase-activated receptor-2 (PAR(2)) is expressed by different types of human leukocytes and involved in the development of inflammatory and infectious diseases. However, its precise role in the regulation of human monocyte and macrophage function during viral infection remains unclear. Also, the ability of PAR(2) agonists to enhance the effects induced by immune mediators during infection or inflammation is still poorly investigated. Therefore, we investigated the ability of a PAR(2) agonist to enhance IFN-gamma-induced suppression of influenza A virus replication in human monocytes. We found that this effect correlates with an increased abundance of IkappaBalpha after costimulation of cells with PAR(2) agonist and IFN-gamma. Remarkably, coapplication of PAR(2) agonist and IFN-gamma also enhances the effects of IFN-gamma on IFN-gamma-inducible protein 10 kDa release, and CD64 and alphaVbeta3 surface expression by human monocytes. Together, these findings indicate a potentially protective role of PAR(2) activation during the progression of influenza A virus infection. This effect could be associated with the ability of PAR(2) agonists to enhance IFN-gamma-induced protective effects on human monocytes.

Agonists of proteinase-activated receptor-2 affect transendothelial migration and apoptosis of human neutrophils.

Skin is the first barrier preventing microorganism invasion in host. Wounds destroy this defense barrier and, without an appropriate care, may lead to sepsis. Neutrophil activation and immigration plays an important role at the inflammatory stage of wound healing. Neutrophils are known to express proteinase-activated receptors (PARs), which can be activated by serine proteases, also by enzymes involved in wound healing. We previously reported that PAR(2) agonists up-regulate cell adhesion molecule expression and cytokine production by human neutrophils. Here, we demonstrate that PAR(2) agonists (serine proteases as well as synthetic peptides) reduce transendothelial migration of neutrophils and prolong their life in vitro. Synthetic PAR(2) agonist also enhanced protective interferon (IFN)gamma-induced FcgammaRI expression at neutrophil cell surface. Of note, IFNgamma is a cytokine, which was used in clinical trials to reactivate human neutrophil functions during sepsis. Moreover, we observed a significant increase of PAR(2) expression on cell surface of neutrophils from septic patients as compared with healthy volunteers. Together, our results indicate that PAR(2) may be involved in the pathophysiology of neutrophil-endothelial interactions during wound healing or later during sepsis in humans, potentially by affecting neutrophil apoptosis, transendothelial migration and Fcgamma receptor-mediated phagocytosis.