Entwicklung neuer Methoden für kompartimentspezifische Analysen des Mikrobioms von Haarfollikeln und assoziierten Entzündungsmediatoren

Background: Basic scientific studies on the regulatory effects of microbial colonization on immune cell responses suggest that hair follicle openings provide a unique niche for continuous exchange with the cutaneous immune system. While the presence of microbiota in the upper follicle is acknowledged, recent bacterial findings at the dermal level encourage a closer look at lower compartments encompassing the bulge and the bulb - two structures featuring a relative immune-privileged status that is indispensable for physiological cycling. Yet, further investigations on host-microbiome interactions in health and disease strongly depend on the availability of in vivo material, indicating a high need for low-invasive sampling methods. Objective: The aim was to establish protocols for extraction and sequencing of bacterial DNA from infrainfundibular compartments of anagen hair follicles, and to add biomarker analyses in order to establish a clinical study set-up suitable for larger studies on patients. Methods: 16S rRNA sequencing was performed on DNA extracts enriched for bacterial DNA from anagen hair follicles plucked from frontal and occipital sites of 12 healthy volunteers (6 females, 6 males) and complemented by fluorescence in situ hybridization (FISH). Based on immunohistochemical stainings for a range of immune cells and inflammatory markers, IL-17A and human β-defensin 2 (HBD2) were selected for confirmation by ELISA on protein extracts from plucked hair follicles. Standard swab analyses and non-invasive readouts including in vivo fluorescence imaging of porphyrins as readout for Propionibacterium (Cutibacterium), scalp surface pH and sebum measurements were added. Results: Enrichment for bacterial DNA from plucked hair follicles gave reproducible results, even though the material was obtained from infrainfundibular portions. Lawsonella clevelandensis, Staphylococcaceae and Propionibacteriaceae were identified as the most abundant bacteria. Accordingly, FISH captured biofilm structures formed by Cutibacterium acnes (formerly Propionibacterium acnes) and Staphylococcus sp. colonies below the infundibulum, although a distinct gradient towards minimal traces in very deep compartments around the bulb was observed. Immunohistochemistry and ELISA revealed the presence of IL-17A, and even more distinctly, of HBD2 in deeper portions of the hair follicles. Conclusion: Hair pulling may be an alternative method to scalp biopsies. Its low invasiveness may help to collect material from larger study groups and even repetitively from the same individual. Reproducible isolation of bacterial DNA from infrainfundibular portions indicates that colonization may reach deeper than expected. A causal relationship to defense molecules like HBD2 remains to be established, but the presence of microbiota in proximity to vital follicular structures may be of relevance for physiological follicular cycling and may also influence the course of some chronic inflammatory hair diseases.Hintergrund: Untersuchungen zu regulatorischen Effekten mikrobieller Kolonisation weisen darauf hin, dass Haarfollikel wichtige Nischen für einen Austausch mit dem Immunsystem der Haut sind. Während Besiedlung des oberflächlich gelegenen Haarfollikelinfundibulums weitreichend beschrieben ist, bilden Nachweise bakteriellen Materials auf der Ebene der Dermis die Grundlage für diese Arbeit. Im Fokus stehen die Wulstregion und der Bulbus, deren immun-priviligierter Status Voraussetzung für einen regulären Haarzyklus ist. Aufgrund der Komplexität sind Möglichkeiten zur nicht-invasiven Gewinnung von repräsentativem in vivo Material eine wesentliche Voraussetzung für weitergehende Untersuchungen. Ziel: Ziel dieser Arbeit war daher die Entwicklung von Protokollen zur Probengewinnung für zukünftige klinische Studien an Patienten. Es ging insbesondere um die Extraktion und Sequenzierung von bakterieller DNA aus infra-infundibulären Kompartimenten von Anagenhaarfollikeln der Kopfhaut und ergänzende Biomarker-Analysen. Methoden: 16S rRNA Sequenzierung erfolgte nach Anreicherung von bakterieller DNA aus Anagenhaarfollikeln, welche zwölf gesunden Probanden (6 weiblich, 6 männlich) sowohl frontal als auch okzipital durch Zupfen von Haarwurzeln entnommen wurden. Ergänzend erfolgte Fluoreszenz in situ Hybridisierung (FISH) an Schnitten von gezogenen Haarwurzeln. Basierend auf immunhistochemischen Färbungen von Immunzellen und Entzündungsmarkern erfolgten ELISA Quantifizierungen für IL-17A und humanes beta-Defensin 2 (HBD2) aus Proteinextrakten. Zusätzlich wurden Standard-Abstrichuntersuchungen und nicht-invasive Messmethoden, darunter Fluoreszenzfotografie als Maß für die Besiedlung mit Propionibacterium (Cutibacterium), pH- und Sebum-Messungen, durchgeführt. Ergebnisse: Obwohl es sich um infra-infundibuläre Kompartimente handelt, konnte bakterielle DNA reproduzierbar aus gezogenen Haarfollikeln angereichert werden. Lawsonella clevelandensis, Staphylococcaceae und Propionibacteriaceae wurden als häufigste Stämme identifiziert. Mittels FISH konnten Biofilme von Cutibacterium acnes (früher: Propionibacterium acnes) und Staphylococcus sp. Kolonien nachgewiesen werden, auch wenn diese zur Tiefe hin deutlich abnahmen. Immunohistochemie und ELISA zeigten IL17A, und noch deutlicher HBD2 in tiefen Kompartimenten. Schlussfolgerung: Das Ziehen von Haarwurzeln kann eine Alternative zur Kopfhautbiopsie sein. Das Vorgehen ist kaum invasiv und kann auch wiederholt am selben Individuum durchgeführt werden kann. Die reproduzierbare Isolierung von bakterieller DNA aus infra-infundibulären Anteilen deutet darauf hin, dass Kolonisierung tiefer reicht als weithin angenommen. Auch wenn die Daten zur Expression von anti-mikrobiellen Peptiden wie HBD2 keinen kausalen Zusammenhang beweisen, könnten Störungen der mikrobiellen Besiedlung und Antworten des daruntergelegenen Gewebes Einfluss auf Haarwuchs und den Verlauf einiger chronisch entzündlicher Haarerkrankungen haben

Stevens-Johnson syndrome induced by combined treatment: carbamazepine and cranial radiation therapy. A case of EMDART?

Introduction . In 1988, Delattre et al. described the first case of erythema multiforme associated with phenytoin and cranial radiation therapy. In 2004, Ahmed et al. coined the term EMPACT syndrome (Erythema Multiforme associated with Phenytoin And Cranial Radiation Therapy). Case report. A 61-year-old patient with glioblastoma was admitted to our hospital with mucosal and cutaneous involvement diagnosed as Stevens-Johnson syndrome induced by combined treatment consisting of carbamazepine and radiation therapy. The cutaneous and mucosal lesions were successfully treated with prednisone and discontinuation of carbamazepine, chemotherapy and radiation therapy. Conclusions. A discussion is ongoing in the literature about a possible association between radiation therapy, anticonvulsant treatment and Stevens-Johnson syndrome. We present a rare case of Stevens-Johnson syndrome induced by carbamazepine combined with radiation therapy and suggest a new acronym – EMDART (Erythema Multiforme associated with Drug And Radiation Therapy) for the description of this entity, as a wider term compared to EMPACT. The newly suggested term includes all diseases in the spectrum of erythema multiforme (also Stevens-Johnson syndrome and toxic epidermal necrolysis) induced by all drugs (not only phenytoin) in association with radiation therapy in all locations

The Potential Relevance of the Microbiome to Hair Physiology and Regeneration: The Emerging Role of Metagenomics

Human skin and hair follicles are recognized sites of microbial colonization. These microbiota help regulate host immune mechanisms via an interplay between microbes and immune cells, influencing homeostasis and inflammation. Bacteria affect immune responses by controlling the local inflammatory milieu, the breakdown of which can result in chronic inflammatory disorders. Follicular microbiome shifts described in some inflammatory cutaneous diseases suggest a link between their development or perpetuation and dysbiosis. Though the hair follicle infundibulum is an area of intense immunological interactions, bulb and bulge regions represent immune-privileged niches. Immune privilege maintenance seems essential for hair growth and regeneration, as collapse and inflammation characterize inflammatory hair disorders like alopecia areata and primary cicatricial alopecia. Current research largely focuses on immunological aberrations. However, studies suggest that external stimuli and interactions across the follicular epithelium can have profound effects on the local immune system, homeostasis, and cycling. Herein, we review hair follicle bacterial colonization, its possible effects on the underlying tissue, and links to the pathogenesis of alopecia, beyond the pure investigation of specific species abundance. As skin microbiology enters the metagenomics era, multi-dimensional approaches will enable a new level of investigations on the effects of microorganisms and metabolism on host tissue

Dysbiosis and Enhanced Beta-Defensin Production in Hair Follicles of Patients with Lichen Planopilaris and Frontal Fibrosing Alopecia

Despite their distinct clinical manifestation, frontal fibrosing alopecia (FFA) and lichen planopilaris (LPP) display similar histopathologic features. Aberrant innate immune responses to endogenous or exogenous triggers have been discussed as factors that could drive inflammatory cascades and the collapse of the stem cell niche. In this exploratory study, we investigate the bacterial composition of scalp skin and plucked hair follicles (HF) of patients with FFA, LPP and alopecia areata circumscripta (AAc), as well as healthy individuals, in relation to cellular infiltrates and the expression of defense mediators. The most abundant genus in lesional and non-lesional HFs of LPP and FFA patients was Staphylococcus, while Lawsonella dominated in healthy individuals and in AAc patients. We observed statistically significant differences in the ratio of Firmicutes to Actinobacteria between healthy scalp, lesional, and non-lesional sites of FFA and LPP patients. This marked dysbiosis in FFA and LPP in compartments close to the bulge was associated with increased HβD1 and HβD2 expression along the HFs from lesional sites, while IL-17A was increased in lesional HF from AAc patients. The data encourage further studies on how exogenous factors and molecular interactions across the HF epithelium could contribute to disease onset and propagation

Dysbiosis and Enhanced Beta-Defensin Production in Hair Follicles of Patients with Lichen Planopilaris and Frontal Fibrosing Alopecia

Despite their distinct clinical manifestation, frontal fibrosing alopecia (FFA) and lichen planopilaris (LPP) display similar histopathologic features. Aberrant innate immune responses to endogenous or exogenous triggers have been discussed as factors that could drive inflammatory cascades and the collapse of the stem cell niche. In this exploratory study, we investigate the bacterial composition of scalp skin and plucked hair follicles (HF) of patients with FFA, LPP and alopecia areata circumscripta (AAc), as well as healthy individuals, in relation to cellular infiltrates and the expression of defense mediators. The most abundant genus in lesional and non-lesional HFs of LPP and FFA patients was Staphylococcus, while Lawsonella dominated in healthy individuals and in AAc patients. We observed statistically significant differences in the ratio of Firmicutes to Actinobacteria between healthy scalp, lesional, and non-lesional sites of FFA and LPP patients. This marked dysbiosis in FFA and LPP in compartments close to the bulge was associated with increased HβD1 and HβD2 expression along the HFs from lesional sites, while IL-17A was increased in lesional HF from AAc patients. The data encourage further studies on how exogenous factors and molecular interactions across the HF epithelium could contribute to disease onset and propagation