The stimulation of TH2 cells results in increased IL-5 and IL-13 production via the H4 receptor.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting in decreased quality of life. Histamine and specifically the H4 receptor play a key role in the inflammatory process in AD and serve as targets for novel therapeutic approaches. OBJECTIVE: In the present study we aimed to elucidate the immunopathological mechanisms with which the H4 receptor impacts TH2 cells and contributes to AD pathophysiology. METHODS: Total CD4+ T cells obtained from healthy or AD individuals and in vitro differentiated TH2 cells were cultured under different conditions and the mRNA expression or protein production of target molecules were determined using quantitative real-time PCR and ELISA. RESULTS: H4 receptor mRNA expression was upregulated concentration dependent upon IL-4 stimulation in in vitro differentiated TH2 cells progressively during the differentiation. Transcriptomic analysis of in vitro differentiated TH2 versus TH1 cells revealed that the H4 receptor among other genes represents one of the highly upregulated genes in TH2 cells. Most importantly, increased amounts of IL-5 and IL-13 mRNA expression were detected in in vitro differentiated TH2 cells as well as protein secretion in the presence of histamine or of the H4 receptor-selective-agonist when compared to the untreated control. CONCLUSION: We show for the first time an H4 receptor dependent upregulation of the pro-inflammatory cytokines IL-5 and IL-13 in human TH2 cells by histamine. This suggests that the blockade of the H4 receptor may lead to downregulation of these cytokines and amelioration of AD symptoms as reported in first clinical studies.

The effect of mogamulizumab on the aberrant T cell population in the peripheral blood - A monocentric retrospective analysis.

BACKGROUND AND OBJECTIVES: The effect of mogamulizumab in cutaneous T-cell lymphoma (CTCL) on T cells (TC) in the peripheral blood and its potential role to navigate treatment intervals are explored. METHODS: We investigated within a retrospective monocentric analysis the effect of mogamulizumab on the CD3+ TC and the aberrant T cell population (TCP), i.e., the CD4+ /CD7- and the CD4+ /CD26- TC, analyzed by flow cytometry. RESULTS: Thirteen patients with CTCL were included. After four cycles there was a mean reduction of 57% in CD3+ TC, 72% in the CD4+ /CD7- and 75% in the CD4+ /CD26- TCP compared to the individual baseline of each patient. The reduction in CD4+ /CD7+ and CD4+ /CD26+ TC was lower, averaging 54% and 41%. A significant decrease in aberrant TCP was already evident after the first administration. A median plateau of TCP already occurred during the IP. Progressive disease occurred in 5/13 patients without a clear correlation to aberrant TCP. CONCLUSIONS: Already after one dose of mogamulizumab, aberrant TCP and, to a lesser extent, normal TC decrease. We did not observe a clear correlation between TCP and the efficacy of mogamulizumab, but further studies with larger numbers of patients are needed.

Histamine Activates Human Eosinophils via H2R and H4R Predominantly in Atopic Dermatitis Patients.

Atopic dermatitis (AD) is maintained by a variety of cells and inflammatory mediators, including eosinophils and histamine. We recently reported that eosinophils from AD patients highly express the H4R. However, its immunomodulatory function in eosinophils is still largely unexplored. In this study, transcriptome analysis of blood eosinophils from AD patients stimulated with histamine and the H4R agonist ST-1006 revealed several regulated genes (e.g., IL-18R, IL-1RL1, PDE4B, CXCR4) involved in inflammation. Subsequently, the impact of histamine on one of the strongly regulated genes, the IL-18 receptor (IL-18Rα), was investigated in detail. Stimulation with histamine induced the upregulation of IL-18Rα at mRNA and at the protein level in human eosinophils, which was more pronounced in cells from AD patients than in cells from healthy controls. IL-18 was upregulated via histamine as well. After pre-incubation with histamine and IFN-γ, subsequent stimulation with IL-18 resulted in an increased ECP mRNA expression. The activation of eosinophils by histamine, in combination with IFN-γ and IL-5, was also accompanied by an upregulation of CD69. Thus, our results indicate a crucial role of histamine in the upregulation of the IL-18/IL-18R axis and in the activation of human eosinophils from AD patients.

Histamine Increases Th2 Cytokine-Induced CCL18 Expression in Human M2 Macrophages.

The chemokine CCL18 is produced in cells of the myelomonocytic lineage and represents one of the most highly expressed chemokines in lesional skin and serum of atopic dermatitis patients. We investigated the role of histamine in CCL18 production in human monocyte-derived M2 macrophages differentiated in the presence of M-CSF and activated with IL-4, IL-13 or with IL-10. Since expression and regulation of histamine H1 receptor (H1R), H2R and H4R by IL-4 and IL-13 on human M2 macrophages were described, we analyzed expression of the histamine receptors in response to IL-10 stimulation by quantitative RT-PCR. IL-10 upregulated H2R and downregulated H4R mRNA expression by trend in M2 macrophages. IL-10, but in a more pronounced manner, IL-4 and IL-13, also upregulated CCL18. Histamine increased the cytokine-induced upregulation of CCL18 mRNA expression by stimulating the H2R. This effect was stronger in IL-10-stimulated M2 macrophages where the upregulation of CCL18 was confirmed at the protein level by ELISA using selective histamine receptor agonist and antagonists. The histamine-induced CCL18 upregulation in IL-10-activated M2 macrophages was almost similar in cells obtained from atopic dermatitis patients compared to cells from healthy control persons. In summary, our data stress a new function of histamine showing upregulation of the Th2 cells attracting chemokine CCL18 in human, activated M2 macrophages. This may have an impact on the course of atopic dermatitis and for the development of new therapeutic interventions.

Histamine 2 Receptor Agonism and Histamine 4 Receptor Antagonism Ameliorate Inflammation in a Model of Psoriasis.

Psoriasis is a chronic inflammatory skin disorder characterized by hyperproliferative keratinocytes and immune cell infiltration into the skin, often accompanied by itch. Histamine, acting via histamine 1-4 receptors, is known to modulate immune responses in the skin and to induce itch. The aim of this study was to test the role of histamine 2 receptors and histamine 4 receptors in the imiquimod-induced psoriasis-like skin inflammation model. BALB/c mice were treated intraperitoneally with amthamine (histamine 2 receptor agonist), JNJ-39758979 (histamine 4 receptor antagonist), a combination of both, or vehicle twice daily in a preventive manner. Imiquimod was applied once daily onto the back skin for 10 consecutive days. Stimulation of histamine 2 receptors and blockade of histamine 4 receptors ameliorated imiquimod-induced skin inflammation. The combination of amthamine and JNJ-39758979 reduced skin inflammation even more pronounced, diminished epidermal hyperproliferation, and inhibited spontaneous scratching behaviour. A combination of histamine 2 receptor agonist and histamine 4 receptor antagonists could represent a new strategy for the treatment of psoriasis.

Anti-tumor effects of tirbanibulin in squamous cell carcinoma cells are mediated via disruption of tubulin-polymerization.

Topical tirbanibulin is a highly effective and well tolerated novel treatment option for actinic keratoses (AKs). This study aimed to characterize the mode of action of tirbanibulin in keratinocytes (NHEK) and cutaneous squamous cell carcinoma (cSCC) cell lines (A431, SCC-12) in vitro. Tirbanibulin significantly reduced proliferation in a dose-dependent manner in all investigated cell lines, inhibited migration, and induced G2/M-cell cycle arrest only in the cSCC cell lines analyzed, and induced apoptosis solely in A431, which showed the highest sensitivity to tirbanibulin. In general, we detected low basal expression of phosphorylated SRC in all cell lines analyzed, therefore, interference with SRC signaling does not appear to be the driving force regarding the observed effects of tirbanibulin. The most prominent tirbanibulin-mediated effect was on ß-tubulin-polymerization, which was especially impaired in A431. Additionally, tirbanibulin induced an increase of the proinflammatory cytokines IL-1α, bFGF and VEGF in A431. In conclusion, tirbanibulin mediated anti-tumor effects predominantly in A431, while healthy keratinocytes and more dedifferentiated SCC-12 were less influenced. These effects of tirbanibulin are most likely mediated via dysregulation of ß-tubulin-polymerization and may be supported by proinflammatory aspects.

Histamine and Th2 cytokines independently and synergistically upregulate MMP12 expression in human M2 macrophages.

Beyond Th2 cells and various immune cells, M2 macrophages have been identified in lesional skin of atopic dermatitis (AD) indicating their involvement in the disease's underlying mechanisms. MMP12, a matrix-degrading enzyme, which is predominantly produced by macrophages, is increased in skin lesions of AD patients. In this study we investigated the expression of MMP12 mRNA in lesional AD skin at single cell level through RNA sequencing (scRNA-seq) and the expression of MMP12 in M2 macrophages from healthy individuals and AD patients in response to Th2 cytokines and histamine using quantitative PCR and ELISA. Additionally, we analyzed macrophages from dupilumab-treated AD patients using the same methods to assess the influence of Th2 cytokines on MMP12 expression ex-vivo. ScRNA-seq identified macrophages as the primary producers of MMP12 in lesional AD skin. In-vitro, both MMP12 mRNA and protein expression were significantly increased in monocytes during differentiation to M2 macrophages in the presence of histamine, of Th2 cytokines or of Th2 cytokines in combination with histamine. In M2 macrophages obtained from dupilumab-treated AD patients, the upregulation of MMP12 expression by IL-4 and IL-13 was attenuated. Our findings unveil a novel mechanism whereby Th2 cytokines and histamine regulate MMP12 expression, potentially impacting skin barrier homeostasis in AD.

The RANKL inhibitor denosumab in combination with dual checkpoint inhibition is associated with increased CXCL-13 serum concentrations.

BACKGROUND: Recent evidence suggests additional immunomodulatory properties of RANKL inhibition possibly boosting the clinical efficacy of immune checkpoint inhibitors (ICI). METHODS: We conducted a prospective, multicentre clinical trial in unresectable stage IV melanoma patients with bone metastases who received denosumab in parallel with dual ICI (BONEMET) and performed comprehensive immune monitoring at baseline and 4, 12, and 24 weeks after initiation of therapy. Secondary endpoints included tolerability and efficacy. For comparison, biospecimens from melanoma patients treated with dual ICI without denosumab were analyzed accordingly and served as retrospective reference cohort. RESULTS: In both the BONEMET (n = 16) and the reference cohort (n = 18) serum levels of 17 cytokines, including IFNγ were significantly increased after 4 weeks of treatment. Patients who received ICI and denosumab showed a significantly higher increase in serum CXCL-13 and a significant decrease in VEGFc compared with the reference cohort. While no changes in T cell composition were observed at 4 weeks, patients in the BONEMET cohort showed a significant decrease in the peripheral naïve T-cell population and an increase in CD8+ effector cells after 12 weeks. Treatment-related adverse events occurred with comparable frequency (93.8% in the BONEMET cohort versus 83.3% in the reference cohort). 7/16 patients in the BONEMET cohort and 8/18 patients in the reference cohort achieved disease control. CONCLUSION: Denosumab in combination with dual ICI modulates cytokine expression and T-cell composition in peripheral blood. The upregulation of CXCL-13, a key factor for initiating tertiary lymphoid structures, strengthens the hypothesis that denosumab indeed boost immunological effects.

A targetable type III immune response with increase of IL-17A expressing CD4+ T cells is associated with immunotherapy-induced toxicity in melanoma.

Immune checkpoint inhibitors are standard-of-care for the treatment of advanced melanoma, but their use is limited by immune-related adverse events. Proteomic analyses and multiplex cytokine and chemokine assays from serum at baseline and at the adverse event onset indicated aberrant T cell activity with differential expression of type I and III immune signatures. This was in line with the finding of an increase in the proportion of CD4+ T cells with IL-17A expression at the adverse event onset in the peripheral blood using flow cytometry. Multiplex immunohistochemistry and spatial transcriptomics on immunotherapy-induced skin rash and colitis showed an increase in the proportion of CD4+ T cells with IL-17A expression. Anti-IL-17A was administered in two patients with mild myocarditis, colitis and skin rash with resolution of the adverse events. This study highlights the potential role of type III CD4+ T cells in adverse event development and provides proof-of-principle evidence for a clinical trial using anti-IL-17A for treating adverse events.

Sirolimus diminishes the expression of GRO-α (CXCL-1) /CXCR2 axis in human keratinocytes and cutaneous squamous cell carcinoma cells.

BACKGROUND: Organ transplant recipients show a high incidence for the formation of cutaneous squamous cell carcinoma (cSCC), while sirolimus appears to reduce the risk. GRO-α is a chemokine, which is overexpressed in many tumor entities and associated with malignant transformation. However, little is known about the expression and function of GRO-α in human cSCC. OBJECTIVE: Our aim was to investigate the relevance of the GRO-α (CXCL-1)/ CXCR2 axis in human cSCC and the potential impact of sirolimus. METHODS: We analyzed the GRO-α expression in human keratinocytes, different cSCC cell lines as well as cSCC tissue and investigated its effect on cell proliferation and migration. Additionally, we incubated cells with sirolimus and measured the expression of GRO-α and its receptor CXCR2. RESULTS: We showed that both constitutive as well as induced GRO-α expression is higher in in cSCC cell lines compared to keratinocytes and that GRO-α protein is detectable in human cSCC tissue. By GRO-α exposure and shRNA knock down, we identified GRO-α as a driving factor in proliferation and migration. Moreover, in a dermis equivalent GRO-α knocked down cSCC cell lines displayed a reduced capacity in tumor nest formation. Incubation with sirolimus significantly inhibited GRO-α expression in keratinocytes as well as tumor cell lines. Moreover, sirolimus decreased the expression of the corresponding receptor CXCR2. CONCLUSION: Taken together, our results suggest that the GRO-α/CXCR2 axis plays a role in human keratinocyte carcinogenesis and might represent a molecular mechanism for the preventive effect of mTOR inhibitors in cSCC development.

Case Report: Sustained Remission Due to PD-1-Inhibition in a Metastatic Melanoma Patient With Depleted B Cells.

Introduction: Checkpoint-Inhibition (CPI) with PD-1- and PD-L1-inhibitors is a well-established therapy for advanced stage melanoma patients. CPI mainly acts via T-lymphocytes. However, recent literature suggests also a role for B cells modulating its efficacy and tolerability of CPI. Case Report: We report a 48-year-old female patient with metastatic melanoma affecting brain, lung, skin and lymph nodes. A preexisting granulomatosis with polyangiitis was treated with rituximab over five years prior to the diagnosis of melanoma, resulting in a complete depletion of B cells both in peripheral blood as well as the tumor tissue. In the absence of the mutation of the proto-oncogene b-raf, treatment with the PD-1 inhibitor nivolumab was initiated. This therapy was well tolerated and resulted in a deep partial response, which is ongoing for 14+ months. Flow cytometric analysis of peripheral blood mononuclear cells revealed 15% IL-10 producing and 14% CD24 and CD38 double positive regulatory B cells. Conclusion: The exceptional clinical response to nivolumab monotherapy in our patient with depleted B cells sheds a new light on the relevance of B cells in the modulation of immune responses to melanoma. Obviously, B cells were not required for the efficacy of CPI in our patient. Moreover, the depletion of regulatory B cells may have improved efficacy of CPI.

Histamine up-regulates oncostatin M expression in human M1 macrophages.

BACKGROUND AND PURPOSE: Human monocyte-derived M1 macrophages develop in relation to growth factors, bacterial products, and cytokines in a local micro-environment. M1 macrophages produce pro-inflammatory mediators, in particular, oncostatin M (OSM), which is secreted from the cells in response to the active complement component C5a. As C5a also releases histamine from human mast cells and shows immune modulatory functions similar to histamine in regulating expression of the IL-12 cytokine family, we investigated the effects of histamine on OSM expression in human M1 macrophages. EXPERIMENTAL APPROACH: Cytokine expression was analysed by real-time quantitative PCR and elisa techniques. Normal human epidermal keratinocytes were stimulated with supernatants from activated M1 macrophages, and phosphorylation of STAT3 was assessed by flow cytometry. KEY RESULTS: OSM mRNA expression was highly up-regulated by histamine and agonists targeting the histamine H1 H2 , and H4 receptors in human M1 macrophages and by C5a, which was used as control stimulus. Protein levels of OSM and IL-6 were up-regulated by histamine. Supernatants from histamine-stimulated, fully differentiated M1 macrophages were able to phosphorylate STAT3 in normal human epidermal keratinocytes. CONCLUSIONS AND IMPLICATIONS: The up-regulation of OSM expression in response to histamine and C5a shown in this study provides further evidence that histamine and C5a, acting through their GPCRs, have almost equal functional effects in cells of the monocyte lineage. Both mediators OSM and IL-6 have the capability to activate human keratinocytes. This effect may have an influence on the course of inflammatory skin diseases. LINKED ARTICLES: This article is part of a themed section on New Uses for 21st Century. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.3/issuetoc.

Stimulation of histamine H4 receptors increases the production of IL-9 in Th9 polarized cells.

BACKGROUND AND PURPOSE: Th9 cells represent a recently defined subset of CD4+ T-helper cells, characterized by a high production of IL-9. They are found at increased frequency in lesions of atopic dermatitis, where IL-9 is also elevated. As histamine is up-regulated in lesions of inflammatory skin diseases, we investigated the expression profile of histamine receptors and their functional role on Th9 cells. EXPERIMENTAL APPROACH: Naïve CD4+ T-cells were purified from human peripheral blood mononuclear cells, using magnetic beads and further differentiated into Th9 cells. During differentiation, cells were additionally stimulated with histamine receptor agonists or left untreated. Histamine receptor expression as well as IL-9 production was measured. KEY RESULTS: As proof of a successful differentiation, IL-9 production was measured at mRNA and protein level. Expression of mRNA for histamine H1 , H2 and H4 receptors were up-regulated in differentiated Th9 cells compared to Th0 cells, while no mRNA for the H3 receptor was detectable. Stimulation of Th9 cells with histamine significantly up-regulated expression of mRNA and protein for IL-9 . Experiments with specific histamine receptor agonists and antagonists revealed that this up-regulation was mediated by H4 receptors. CONCLUSIONS AND IMPLICATIONS: In summary, our study demonstrates a functional role for histamine H4 receptors on Th9 cells, which might amplify the pro-inflammatory potency of these cells. Together with earlier studies on Th2 and Th17 cells, this study underlines the promising approach for the use of H4 receptor antagonists in inflammatory and allergic diseases such as atopic dermatitis. LINKED ARTICLES: This article is part of a themed section on New Uses for 21st Century. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.3/issuetoc.

The role of the histamine H4 receptor in atopic dermatitis and psoriasis.

Atopic dermatitis (AD) and psoriasis are common skin diseases with a high negative impact on patients' quality of life. Both diseases are mediated by a pro-inflammatory infiltrate consisting of several cell types, such as T-cells, antigen-presenting cells and granulocytes and display disturbed keratinocyte differentiation. Given the fact that histamine levels are also highly elevated in inflamed skin, it is likely that histamine plays a relevant role in disease pathology. However, antagonists blocking histamine H1 receptor or H2 receptors are largely ineffective in reducing chronic symptoms in AD and psoriasis. Over the last years, much research has been undertaken to shed light into the mode of action of the most recently discovered histamine H4 receptor. This research has shown that H4 receptor antagonists display antipruritic and anti-inflammatory effects not only in mouse models but also in first human clinical trials, and therefore, H4 receptors might present a novel therapeutic target. In this review, we summarize the effects of the H4 receptors on different cell types, mouse models and clinical studies in regard to AD and psoriasis respectively. LINKED ARTICLES: This article is part of a themed section on New Uses for 21st Century. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.3/issuetoc.

The mTOR-inhibitor Sirolimus decreases the cyclosporine-induced expression of the oncogene ATF3 in human keratinocytes.

BACKGROUND: Due to their immunosuppressive therapy, organtransplant recipients (OTRs) exhibit a high incidence for the development of cutaneous squamous cell carcinoma (cSCC). Randomized studies of kidney-transplanted patients indicate a significant lower susceptibility for cSCC among patients receiving the mTOR-inhibitor Sirolimus, compared to patients without mTOR-regimen. The exact mechanism, how mTOR inhibition affects keratinocyte carcinogenesis remains unclear. OBJECTIVE: Our aim was to investigate the impact of Sirolimus on the expression level of the oncogene ATF3, which is involved in the development and progression of cSCC. METHODS: We incubated human keratinocytes, cSSC cell lines and 3D skin equivalents with Sirolimus, exposed the cells to calcineurin inhibitors (CNI) and UVA-radiation and measured the expression level of ATF3 by real-time PCR and western blot. RESULTS: We show that Sirolimus downregulates the expression of ATF3 induced by cyclosporine or cyclosporine plus UV-radiation in keratinocytes. In line with this we demonstrate a decrease in ATF3 expression, by incubating 3D skin equivalents with Sirolimus prior to cyclosporine and UV-light. However, Sirolimus has no significant impact on the ATF3 expression levels of cyclosporine stimulated cSCC cell lines. CONCLUSION: Taken together, our study demonstrates that Sirolimus downregulates the CNI or UV-induced ATF3 expression in human keratinocytes, which could be a potential molecular mechanism how Sirolimus reduces cSCC in OTRs. The lack of ATF3 suppression by Sirolimus in cSCC cell lines fits to observations from clinical studies which demonstrated a clinical benefit from the switch to a mTOR-regimen in patients with low tumor burden in early stage of disease.