Nlrp6- and ASC-Dependent Inflammasomes Do Not Shape the Commensal Gut Microbiota Composition.

The gut microbiota regulate susceptibility to multiple human diseases. The Nlrp6-ASC inflammasome is widely regarded as a hallmark host innate immune axis that shapes the gut microbiota composition. This notion stems from studies reporting dysbiosis in mice lacking these inflammasome components when compared with non-littermate wild-type animals. Here, we describe microbial analyses in inflammasome-deficient mice while minimizing non-genetic confounders using littermate-controlled Nlrp6-deficient mice and ex-germ-free littermate-controlled ASC-deficient mice that were all allowed to shape their gut microbiota naturally after birth. Careful microbial phylogenetic analyses of these cohorts failed to reveal regulation of the gut microbiota composition by the Nlrp6- and ASC-dependent inflammasomes. Our results obtained in two geographically separated animal facilities dismiss a generalizable impact of Nlrp6- and ASC-dependent inflammasomes on the composition of the commensal gut microbiota and highlight the necessity for littermate-controlled experimental design in assessing the influence of host immunity on gut microbial ecology.

Histopathologic regression in patients with primary cutaneous melanoma undergoing sentinel lymph node biopsy is associated with favorable survival and, after metastasis, with improved progression-free survival on immune checkpoint inhibitor therapy: A single-institutional cohort study.

BACKGROUND: Histopathologic regression of cutaneous melanoma is considered a favorable prognostic factor, but its significance in clinical practice remains controversial. OBJECTIVE: To investigate the prognostic importance of regression in patients with primary cutaneous melanoma undergoing sentinel lymph node (SLN) biopsy and to assess its significance in patients progressing to an unresectable stage requiring systemic therapy. METHODS: We retrospectively reviewed patients with newly diagnosed melanoma undergoing SLN biopsy between 2010 and 2015 and available information on histopathologic regression (n = 1179). Survival data and associations of clinical variables with SLN status were assessed. RESULTS: Patients with regressive melanoma showed favorable relapse-free (hazard ratio [HR], 0.52; P = .00013), distant metastasis-free (HR, 0.56; P = .0020), and melanoma-specific survival (HR, 0.35; P = .00053). Regression was associated with negative SLN (odds ratio, 0.48; P = .0077). In patients who progressed to an unresectable stage, regression was associated with favorable progression-free survival under immune checkpoint inhibition (HR, 0.43; P = .031) but not under targeted therapy (HR, 1.14; P = .73) or chemotherapy (HR, 3.65; P = .0095). LIMITATIONS: Retrospective, single-institutional design. CONCLUSIONS: Regression of cutaneous melanoma is associated with improved prognosis in patients eligible for SLN biopsy as well as in patients with unresectable disease receiving systemic therapy with immune checkpoint inhibitors.

Human sleep consolidates allergic responses conditioned to the environmental context of an allergen exposure.

Allergies are highly prevalent, and allergic responses can be triggered even in the absence of allergens due to Pavlovian conditioning to a specific cue. Here we show in humans suffering from allergic rhinitis that merely reencountering the environmental context in which an allergen was administered a week earlier is sufficient to trigger an allergic response-but only if participants had slept after allergen exposure. This context-conditioning effect was entirely absent when participants stayed awake the night after allergen exposure or were tested in a different context. Unlike in context conditioning, cue conditioning (to an odor stimulus) occurred independently of sleep, a differential pattern that is likewise observed for conditioning in the behavioral domain. Our findings provide evidence that allergic responses can be conditioned to contextual information alone, even after only a single-trial conditioning procedure, and that sleep is necessary to consolidate this rapidly acquired maladaptive response. The results unravel a mechanism that could explain part of the strong psychological impact on allergic responses.

Non invasive in vivo monitoring of dimethyl fumarate treatment in EAE by assessing the glucose metabolism in secondary lymphoid organs.

[18 F]FDG-PET/CT is a high sensitive functional diagnostic imaging modality to monitor tumor but also immune cell activation by determination of the glucose metabolism. Our results show that the anti-inflammatory effects of immunotherapeutics like DMF can be assessed non invasively in vivo during Th1/Th17 cell-mediated encephalomyelitis (EAE) by [18 F]FDG-PET/CT imaging of the draining lymph nodes.

Immunophenotyping in pemphigus reveals a TH17/TFH17 cell-dominated immune response promoting desmoglein1/3-specific autoantibody production.

BACKGROUND: TH2 cells were thought to be a pivotal factor for initiation of the autoimmune blistering disease pemphigus. However, the role of other T-cell subsets in pemphigus pathogenesis remained unclear. OBJECTIVE: We aimed to characterize the exact phenotype of T cells responsible for the development of pemphigus. METHODS: Whole transcriptome shotgun sequencing was performed to determine differential gene expression in pemphigus lesions and skin of healthy individuals. The cutaneous cytokine signature was further evaluated by real-time quantitative PCR. In peripheral blood, the distribution of TH cell and folliclular helper (TFH) cell subsets was analyzed by flow cytometry. Finally, the capacity of TH and TFH cell subsets to induce desmoglein (Dsg)-specific autoantibodies by memory B cells was evaluated in coculture experiments. RESULTS: Transcriptome analysis of skin samples identified an IL-17A-dominated immune signature in patients with pemphigus, and Kyoto Encyclopedia of Genes and Genomes pathway analysis confirmed the dominance of the IL-17A signaling pathway. Increased expression of IL17A and associated cytokines was also detected by real-time quantitative PCR comparing lesional with perilesional or healthy skin. Interestingly, utilization of flow cytometry showed that patients with active pemphigus had elevated levels of circulating IL-17+, TH17, TFH17, and TFH17.1 cells. Notably, levels of TH17 and TFH17 cells correlated with levels of Dsg-specific CD19+CD27+ memory B cells, and patients with acute pemphigus showed higher levels of Dsg3-autoreactive TFH17 cells. Coculture experiments revealed TFH17 cells as primarily responsible for inducing Dsg-specific autoantibody production by B cells. CONCLUSION: Our findings show that TFH17 cells are critically involved in the pathogenesis of pemphigus and offer novel targets for therapeutic intervention.

Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1.

Through their capacity to sense danger signals and to generate active interleukin-1ß (IL-1ß), inflammasomes occupy a central role in the inflammatory response. In contrast to IL-1ß, little is known about how IL-1α is regulated. We found that all inflammasome activators also induced the secretion of IL-1α, leading to the cosecretion of both IL-1 cytokines. Depending on the type of inflammasome activator, release of IL-1α was inflammasome dependent or independent. Calcium influx induced by the opening of cation channels was sufficient for the inflammasome-independent IL-1α secretion. In both cases, IL-1α was released primarily in a processed form, resulting from intracellular cleavage by calpain-like proteases. Inflammasome-caspase-1-dependent release of IL-1α and IL-1ß was independent of caspase-1 catalytic activity, defining a mode of action for caspase-1. Because inflammasomes contribute to the pathology of numerous chronic inflammatory diseases such as gout and diabetes, IL-1α antagonists may be beneficial in the treatment of these disorders.

Stevens-Johnson syndrome and toxic epidermal necrolysis: a 10-year experience in a burns unit.

OBJECTIVE: Stevens-Johnson syndrome (SJS) and its more severe counterpart, toxic epidermal necrolysis (TEN), are skin hypersensitivity reactions defined by epidermal blistering and necrosis. The exact pathophysiology of SJS/TEN is yet to be deciphered, but a number of risk factors have been identified including adverse drug reactions. The diagnosis of SJS/TEN is made on a clinical basis, and treatment consists of supportive care and occasionally immunosuppressants, such as cyclosporin, high-dose intravenous immunoglobulins and/or corticosteroids. Mortality rates can reach 20-25% in adults but are reduced with early intervention. To identify optimal treatment regimens, to better understand the patient cohort affected, and to help identify key risk factors for mortality, we report our experience with the treatment and management of SJS/TEN patients. METHODS: A retrospective review of consecutive patients with SJS and/or TEN admitted to a single burns centre in Germany, between 2008 and 2018, was conducted. The primary outcomes of demographics, clinical course, treatment and patient-reported outcomes were recorded and compared with a control group of patients with burns without a diagnosis of SJS/TEN. RESULTS: A total of 23 patients with SJS/TEN met the inclusion criteria: 17 (74%) with TEN; four (17%) with SJS/TEN overlap; and two (9%) with SJS. Of the patients, 14 (61%) were female and nine (39%) were male. Patient age ranged from 32-78 years (mean: 52 years). A matched cohort of 23 patients with burns served as the control group. All patients received standard of care with a multidisciplinary team. Compared with the control group, SJS/TEN patients had higher mortality rates (n=6, 26% versus n=8, 35%, respectively). The average age of death was 69 years in SJS/TEN patients versus 63 years in control group patients. Age and SCORTEN scores were significant predictors of mortality. CONCLUSIONS: SJS and TEN are rare but extreme reactions of the skin and mucosa, associated with high disease mortality rates. This 10-year single-centre retrospective review contributes to the bank of information for reviews evaluating the management of SJS/TEN patients.

Stevens-Johnson syndrome and toxic epidermal necrolysis: a systematic review and meta-analysis.

OBJECTIVE: Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare and severe skin and mucosal reactions that are associated with high mortality. Despite the severity, an evidence-based treatment protocol for SJS/TEN is still lacking. METHOD: In this systematic review and meta-analysis, the PubMed database was searched using the following terms: [Stevens-Johnson syndrome] OR [toxic epidermal necrolysis] AND [therapy] OR [treatment] over a 20-year period (1999-2019) in the German and English language. All clinical studies reporting on the treatment of SJS/TEN were included, and epidemiological and diagnostic aspects of treatment were analysed. A meta-analysis was conducted on all comparative clinical studies that met the inclusion criteria. RESULTS: A total of 88 studies met the inclusion criteria, reporting outcomes in 2647 patients. Treatment was either supportive or used systemic corticosteroid, intravenous immunoglobulin, plasmapheresis, cyclosporine, thalidomide or cyclophosphamide therapy. The meta-analysis included 16 (18%) studies, reporting outcomes in 976 (37%) patients. Systemic glucocorticoids showed a survival benefit for SJS/TEN patients in all analyses compared with other forms of treatment. Cyclosporine treatment also showed promising results, despite being used in a small cohort of patients. No beneficial effects on mortality could be demonstrated for intravenous immunoglobulins. CONCLUSION: Glucocorticoids and cyclosporine may be tentatively recommended as the most promising immunomodulatory therapies for SJS/TEN, but these results should be investigated in future prospective controlled trials.

RIPK1 and TRADD Regulate TNF-Induced Signaling and Ripoptosome Formation.

TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.

Molecular Mechanisms of Hair Growth and Regeneration: Current Understanding and Novel Paradigms.

Hair is a defining feature of mammals and has critical functions, including protection, production of sebum, apocrine sweat and pheromones, social and sexual interactions, thermoregulation, and provision of stem cells for skin homeostasis, regeneration, and repair. The hair follicle (HF) is considered a "mini-organ," consisting of intricate and well-organized structures which originate from HF stem and progenitor cells. Dermal papilla cells are the main components of the mesenchymal compartments in the hair bulb and are instrumental in generating signals to regulate the behavior of neighboring epithelial cells during the hair cycle. Mesenchymal-epithelial interactions within the dermal papilla niche drive HF embryonic development as well as the postnatal hair growth and regeneration cycle. This review summarizes the current understanding of HF development, repair, and regeneration, with special focus on cell signaling pathways governing these processes. In particular, we discuss emerging paradigms of molecular signaling governing the dermal papilla-epithelial cellular interactions during hair growth and maintenance and the recent progress made towards tissue engineering of human hair follicles.

TNF Is Partially Required for Cell-Death-Triggered Skin Inflammation upon Acute Loss of cFLIP.

cFLIP is required for epidermal integrity and skin inflammation silencing via protection from TNF-induced keratinocyte apoptosis. Here, we generated and analyzed cFLIP epidermal KO mice with additional TNF deficiency. Intriguingly, the ablation of TNF rescued the pathological phenotype of epidermal cFLIP KO from characteristic weight loss and increased mortality. Moreover, the lack of TNF in these animals strongly reduced and delayed the epidermal hyperkeratosis and the increased apoptosis in keratinocytes. Our data demonstrate that TNF signaling in cFLIP-deficient keratinocytes is the critical factor for the regulation of skin inflammation via modulated cytokine and chemokine expression and, thus, the attraction of immune cells. Our data suggest that autocrine TNF loop activation upon cFLIP deletion is dispensable for T cells, but is critical for neutrophil attraction. Our findings provide evidence for a negative regulatory role of cFLIP for TNF-dependent apoptosis and partially for epidermal inflammation. However, alternative signaling pathways may contribute to the development of the dramatic skin disease upon cFLIP deletion. Our data warrant future studies of the regulatory mechanism controlling the development of skin disease upon cFLIP deficiency and the role of cFLIP/TNF in a number of inflammatory skin diseases, including toxic epidermal necrolysis (TEN).

Postoperative complications in dermatological patients undergoing microscopically controlled surgery in inpatient setting (next-day surgery): A single-center epidemiological study.

BACKGROUND: Surgical site infections (SSI), bleeding, and necrosis are possible complications of dermatological surgery, and their rates are well described for Mohs surgery (same-day surgery). However, there are only limited data on their occurrence in microscopically controlled surgery of the form in which it is practiced in German hospitals (next-day surgery). MATERIALS AND METHODS: We performed a retrospective analysis of patient records of patients hospitalized for microscopically controlled surgery during the year 2017 (12 months) in the Department of Dermatology and Allergology at the University Hospital of the RWTH Aachen (Aachen, Germany). The investigation addressed postoperative outcomes. RESULTS: 319 patients underwent 528 dermatosurgical procedures in the defined period. Bleeding and necrosis occurred in 3.8 % (20/528) and 1.7 % (9/528) of the procedures, respectively. SSI occurred in 5.1 % (27/528) of the cases. The occurrence of bleeding was a statistically significant risk factor for SSI (p  =  0.01). Furthermore, bleeding, SSI, and wound closure with a full-thickness graft were statistically significant risk factors for the development of necrosis (p < 0.05). Diabetes or immunosuppression were not found to be statistically significant risk factors for the development of SSI or necrosis after dermatologic surgery (p > 0.05). CONCLUSIONS: Complication rates in microscopically controlled surgery (next-day surgery) are generally low and similar to those reported for Mohs surgery (same-day surgery). Therefore, it appears that some evidence-based perioperative recommendations that have been developed for Mohs surgery could be applied to German inpatient dermatosurgery. However, prospective studies with larger patient numbers are required to offer concrete recommendations specifically for microscopically controlled surgery (next-day surgery).

Cholesterol Modification of p40-Specific Small Interfering RNA Enables Therapeutic Targeting of Dendritic Cells.

Small interfering RNA (siRNA)-based therapies allow targeted correction of molecular defects in distinct cell populations. Although efficient in multiple cell populations, dendritic cells (DCs) seem to resist siRNA delivery. Using fluorescence labeling and radiolabeling, we show that cholesterol modification enables siRNA uptake by DCs in vitro and in vivo. Delivery of cholesterol-modified p40 siRNA selectively abolished p40 transcription and suppressed TLR-triggered p40 production by DCs. During immunization with peptide in CFA, cholesterol-modified p40 siRNA generated p40-deficient, IL-10-producing DCs that prevented IL-17/Th17 and IFN-γ/Th1 responses. Only cholesterol-modified p40-siRNA established protective immunity against experimental autoimmune encephalomyelitis and suppressed IFN-γ and IL-17 expression by CNS-infiltrating mononuclear cells without inducing regulatory T cells. Because cholesterol-modified siRNA can thus modify selected DC functions in vivo, it is intriguing for targeted immune therapy of allergic, autoimmune, or neoplastic diseases.

A role for mitochondria in NLRP3 inflammasome activation.

An inflammatory response initiated by the NLRP3 inflammasome is triggered by a variety of situations of host 'danger', including infection and metabolic dysregulation. Previous studies suggested that NLRP3 inflammasome activity is negatively regulated by autophagy and positively regulated by reactive oxygen species (ROS) derived from an uncharacterized organelle. Here we show that mitophagy/autophagy blockade leads to the accumulation of damaged, ROS-generating mitochondria, and this in turn activates the NLRP3 inflammasome. Resting NLRP3 localizes to endoplasmic reticulum structures, whereas on inflammasome activation both NLRP3 and its adaptor ASC redistribute to the perinuclear space where they co-localize with endoplasmic reticulum and mitochondria organelle clusters. Notably, both ROS generation and inflammasome activation are suppressed when mitochondrial activity is dysregulated by inhibition of the voltage-dependent anion channel. This indicates that NLRP3 inflammasome senses mitochondrial dysfunction and may explain the frequent association of mitochondrial damage with inflammatory diseases.