Investigation of pulmonary gene expression at the single cell level in (pneumococcal) pneumonia

Hintergrund: Die Lunge ist ein mikroanatomisch komplexes Organ, in dem über 40 verschiedene Zellpopulationen zu dem Erhalt der physiologischen Organfunktion bei-tragen. Während einer akuten bakteriellen Infektion wie der ambulant erworbenen Pneumonie (engl.: community acquired pneumonia, CAP) migrieren weitere Leukozytenpopulationen in die Lunge. Die bakterielle CAP stellt sowohl in Europa als auch weltweit eine häufige und potenziell letale Erkrankung dar, was ein besseres Verständnis der Pathophysiologie mit allen beteiligten Zelltypen erstrebenswert macht. In dieser Arbeit sollte ein Protokoll etabliert und validiert werden, das Einzelzell-Transkriptomanalysen im infektiologischen Kontext ermöglicht, sowie erhaltene Datensätze exploratorisch analysiert werden. Methodik: Drei Zellisolationsprotokolle wurden evaluiert, um Einzelzell-Suspensionen mit hoher Viabilität und Singularität aus murinem Lungengewebe von mit Streptococcus pneumoniae Serotyp 2 (S. pn), der Pneumolysin-defizienten Pneumokokken-Mutante (S. pn Δply) oder mit Lösungsmittel (Kontrollgruppe) infizierten Versuchstieren zu isolieren. Nachfolgend wurde eine magnetische Zell-Aufreinigung durchgeführt, Genexpressionsbibliotheken erstellt und diese sequenziert. Die Datenauswertung erfolgte durch Software des Herstellers sowie R und entsprechende Pakete. Ergebnisse: Das erhaltene Transkriptom von 10.933 Zellen konnte Leukozyten, Epithelzellen, Endothelzellen und mesenchymalen Zellen zugeordnet werden, eine übermäßige Expression an Stressgenen wurde vermieden. Fibroblasten zeigten eine starke Reaktion durch die Regulation der Gene Lcn2 und Gsn nach Infektion mit S. pn. Makrophagen exprimierten nach Infektion, in Abhängigkeit vom Virulenzfaktor Pneumolysin, ein Muster an Genen des Interferon-Signalwegs. Granulozyten zeigten insbesondere eine Cxcl2 Hochregulierung sowie eine verminderte Expression von Ccl3, Cxcl3 und Il1rn durch Pneumolysin. Schließlich war die Anzahl an Zell-Interaktionen verschiedener Zelltypen durch inflammatorische Geschehnisse reduziert. Schlussfolgerung: Die Möglichkeit, unterschiedliche Zellpopulationen auf Einzelzell-ebene zu untersuchen, erweitert insbesondere in der Lunge als heterogenem Organ das Verständnis von pathophysiologischen Geschehnissen. Wenn die Ergebnisse mit anderen Methoden vergleichbarer Auflösung wie der Proteomik, Sekretom-Analysen oder Spatial transcriptomics validiert werden, kann dies den Weg zu neuen Therapieansätzen ebnen.Background: The lung is a micro-anatomically complex organ harboring more than 40 different cell populations essential for maintaining physiological organ function. During an acute bacterial infection, such as community acquired pneumonia (CAP), leukocyte populations migrate to the lung further expanding the cellular landscape. Bacterial CAP is a common and potentially lethal disease both in Europe and worldwide, and therefore a better understanding of the pathophysiology and cell types involved is crucial. The aim of this study was to establish and validate a protocol that is applicable for single-cell transcriptome analysis in infectious disease research and to perform exploratory analysis on the generated data sets. Methods: Three cell isolation protocols were evaluated to ensure technical and qualitative requirements of isolating single cell suspensions with high viability and singularity using murine lung tissue. Mice were infected with Streptococcus pneumoniae serotype 2 (S. pn), the pneumolysin-deficient pneumococcal mutant (S. pn Δply) or solvent (control group), followed by magnetic cell sorting, gene expression library construction and sequencing. Data analysis was performed using the manufacturer’s software and R, including add-on packages for single-cell analysis. Results: The resulting transcriptome of 10,933 cells could be assigned to leukocytes, epithelial cells, endothelial cells, and mesenchymal cells while excessive expression of stress genes was avoided. Fibroblasts showed a strong response to infection with S. pn through regulation of the genes Lcn2 and Gsn. Macrophages expressed a pattern of interferon pathway genes after infection, dependent on the presence of the virulence factor pneumolysin. Granulocytes exhibited upregulation of Cxcl2 and downregulation of Ccl3, Cxcl3 and Il1rn by pneumolysin. Finally, the number of cell-cell interactions of different origin was reduced by inflammatory events. Conclusion: The possibility of studying different cell populations at a single-cell level may contribute to a better understanding of pulmonary pathophysiological events, especially considering the very heterogeneous nature of the lungs. Thus, single-cell RNA-sequencing could pave the way to new therapeutic approaches, particularly if results are validated with other methods of comparable resolution such as proteomics, secretomics or spatial transcriptomics

Systematic review and meta-analysis of the efficacy and safety of self-applied topical interventions for melasma

Hintergrund Das Melasma ist eine verbreitete dermatologische Erkrankung, die aufgrund ihrer Prädilektionsstellen im Bereich der sichtbaren Haut das emotionale Wohlbefinden und die allgemeine Lebensqualität der Betroffenen einschränken kann. Ein breites Spektrum an medizinischen, kosmetischen und pflanzlichen Therapieoptionen, bei nach dem heutigen Stand uneinheitlichen Ergebnissen und häufigen Rückfällen, erschwert die Auswahl der am besten geeigneten Behandlung. Ziel Ziel war es, randomisierte, kontrollierte, verblindete Studien zur topischen, selbstapplizierten Therapie bei Melasma systematisch auszuwerten und kritisch zu beurteilen. Methodik Eine systematische Übersichtsarbeit und Metaanalyse wurden im Einklang mit der Cochrane-Methodik und den Empfehlungen der Arbeitsgruppe GRADE erstellt. Es erfolgte eine systematische Literaturrecherche der elektronischen Datenbanken MEDLINE und Cochrane CENTRAL. Um eingeschlossen zu werden, mussten die folgenden Einschlusskriterien erfüllt sein: Eingeschlossen wurden randomisierte, kontrollierte Studien (RCT) mit mindestens 15 Teilnehmer*innen pro Studienarm, in denen ausdrücklich die Methode zur Erstellung der Randomisierungssequenz angegeben wurde und die Ergebnisbewertung verblindet war. Die Daten eingeschlossener Studien wurden, sofern möglich, gepoolt und metaanalysiert. Resultate Die systematische Literaturrecherche erzielte 1078 Treffer, von denen 36 Studien ein-geschlossen wurden. Die 47 berichteten Vergleiche von Interventionen zur Behandlung des Melasmas umfassen medizinische, kosmetische und pflanzliche Therapieoptionen. Für eine Vielzahl von Interventionen waren Direktvergleiche verfügbar, deren Ergebnisse jedoch heterogen waren. Ein Pooling von Daten war möglich für die Vergleiche von Tranexamsäure vs. Hydrochinon und Cysteamin vs. Placebo. Das Vertrauen in die Effektschätzer reichte von sehr gering bis hoch. Schlussfolgerung Die Ergebnisse zeigen, dass die etablierten Therapieoptionen Dreifachkombinationscreme, bestehend aus Fluocinolonacetonid, Hydrochinon und Tretinoin, sowie ihre Einzelkomponenten Hydrochinon und Tretinoin zu einer Aufhellung des Melasmas führen. Es gibt Hinweise, dass die Verwendung eines Breitspektrum-Sonnenschutzmittels, das sowohl das ultraviolette als auch das sichtbare Lichtspektrum abdeckt, die Wirksamkeit der Behandlung mit Hydrochinon erhöht. Wir konnten weitere vielversprechende medizinische, kosmetische und pflanzliche Behandlungsansätze identifizieren. Es sind weitere RCTs erforderlich, in denen die etablierten topischen Therapien verglichen werden, sowie Studien, die deren langfristige Wirksamkeit untersuchen, um unser Wissen über die geeignete Therapie zu erweitern.Background Melasma is a common dermatologic condition that can influence the emotional well-being and overall quality of life of patients due to affecting the visible skin. A broad range of medical, cosmetic, and herbal treatment options, characterized by inconsistent results and frequent relapses is available, making it difficult to select the most appropriate treatment. Objectives We systematically evaluated and critically assessed randomized, investigator-blinded clinical trials on topical self-applied interventions for melasma. Methods A systematic review and meta-analysis were performed in accordance with the Cochrane methodology and the GRADE working group recommendations. A systematic literature search of the MEDLINE and Cochrane CENTRAL electronic databases was performed. For the studies to be included, the following inclusion criteria had to be met: Randomized controlled trials (RCTs) with at least 15 participants per study arm, which explicitly stated the method for generating the random allocation sequence and that the outcome assessment was blinded, were included. Data from included studies were pooled and meta-analysed whenever possible. Results The systematic literature search yielded 1078 hits, of which 36 studies were included. The 47 reported comparisons of interventions for the treatment of melasma included medical, cosmetic, and herbal therapy options. Direct comparisons were available for a variety of interventions, but results were heterogeneous. Pooling of data was possible for the comparisons tranexamic acid (TXA) vs. hydroquinone (HQ) and cysteamine vs. placebo. Confidence in the effect estimates ranged from very low to high. Conclusion The results show that the established therapeutic options triple combination cream (TCC), consisting of fluocinolone acetonide, hydroquinone and tretinoin, and its individual components HQ and tretinoin lead to effective lightening of melasma. There is evidence that the use of a broad-spectrum sunscreen covering both the visible and ultraviolet light spectra increases the efficacy of treatment with HQ. We identified other promising medical, cosmetic, and herbal treatment approaches. Further RCTs comparing these established topical therapies are needed in the future, as well as studies investigating their long-term efficacy to increase our knowledge of appropriate therapy

Self-applied topical interventions for melasma: a systematic review and meta-analysis of data from randomized, investigator-blinded clinical trials

Background Melasma is a common dermatological condition. Although its relevance as a skin condition is primarily of a cosmetic nature, it may affect the patient’s wellbeing and quality of life. A broad range of treatment options is available, which makes it difficult to choose the most appropriate of those treatments. Objectives To summarize and critically appraise evidence from investigator‐blinded randomized controlled trials (RCTs) on the efficacy and safety of self‐applied topical interventions for melasma. Methods We systematically searched MEDLINE and the Cochrane CENTRAL trials database for RCTs on topical, self‐administered interventions for patients diagnosed with melasma. Eligibility was limited to RCTs that explicitly stated in their methods section (i) how they generated the random allocation sequence, and (ii) that the study outcome assessor was blinded to the participants’ group allocation. Outcomes of interest included evaluator‐assessed clinical scores (such as the Melasma Area and Severity Index), quality of life and patient‐reported outcomes, as well as safety outcomes. The study findings were meta‐analysed, pooling data from studies on the same comparisons, if this was possible. We assessed confidence in effect estimates using the GRADE approach. Results Our searches yielded 1078 hits. We included 36 studies reporting on 47 different comparisons of interventions. These included medical treatments such as ‘triple combination cream’ (TCC), over‐the‐counter cosmetic and herbal products, as well as sun creams covering different light spectra. Pooling data was possible for only two comparisons, topical tranexamic acid (TXA) vs. hydroquinone (HQ) and cysteamine vs. placebo. Direct comparisons were available for a variety of interventions; however, the reported outcomes varied greatly. Overall, our confidence in the effect estimates ranged from very low to high. Conclusions Our findings indicate that TCC and its individual components HQ and tretinoin are effective in lightening melasma. Besides these established self‐applied treatment options, we identified further medical treatments as well as promising cosmetic and herbal product treatment approaches. Furthermore, evidence suggests that using broad‐spectrum sunscreen covering both the visible and ultraviolet‐light spectrum enhances the treatment efficacy of HQ. However, with mostly small RCTs comparing treatments directly using a broad range of outcomes, further research is needed to draw conclusions about which treatment is most effective

Impact of off‐label use regulations on patient care in dermatology – a prospective study of cost‐coverage applications filed by tertiary dermatology clinics throughout Germany

Background In dermatology, a medical speciality with a relatively high number of rare diseases, physicians often have to resort to off-label treatment options. To avoid claims, physicians in Germany can file a cost-coverage request (off-label application, OL-A). Objectives Our aim was to investigate the extent to which the current regulations affect patient care. Material and methods Prospective cohort study among tertiary dermatology clinics throughout Germany, consecutively including OL-As (05/2019–09/2020) and assessing the follow-up correspondence. We modelled regressions to assess factors associated with cost-coverage decisions and the time needed by health insurers to process the OL-As. Results Thirteen clinics provided data on 121 OL-As, two of which applied for on-label treatments. Of the remaining 119 OL-As, 70 (58.8%) were immediately approved and 44 (37.0%) rejected. Including cases with one or more appeals, 87 of 119 OL-As (73.1%) were finally approved and 26 (21.9%) rejected. There was an association of the final approval rate with (1) the class of medication/treatment, with approval rates being significantly lower for JAK inhibitors than for biologics (OR 0.16, 95%-CI: 0.03–0.82); (2) German state, with approval rates being lower in eastern than in western states (OR 0.30, 95%-CI 0.12–0.76); and (3) cost of the intervention (no linear trend). However, none of these predictors was significant in our multiple logistic regression models. The median health insurer's processing time (first response) was 29 days (IQR 22–38). Our analyses showed no evidence of an association with the predictors we assessed. In cases approved, the median time from the decision to file an OL-A to the actual initiation of the treatment was 65.5 days (IQR 51–92). Conclusions Our study points to substantial delays and inequalities in the provision of timely health care for dermatological patients with rare diseases, often involving treatments for which there is no adequate approved therapy

Protocol to dissociate healthy and infected murine- and hamster-derived lung tissue for single-cell transcriptome analysis

In infectious disease research, single-cell RNA sequencing allows dissection of host-pathogen interactions. As a prerequisite, we provide a protocol to transform solid and complex organs such as lungs into representative diverse, viable single-cell suspensions. Our protocol describes performance of vascular perfusion, pneumonectomy, enzymatic digestion, and mechanical dissociation of lung tissue, as well as red blood cell lysis and counting of isolated cells. A challenge remains, however, to further increase the proportion of pulmonary endothelial cells without compromising on viability. For complete details on the use and execution of this protocol, please refer to Nouailles et al. (2021), Wyler et al. (2022), and Ebenig et al. (2022)

MicroRNA-223 Dampens Pulmonary Inflammation during Pneumococcal Pneumonia

Community-acquired pneumonia remains a major contributor to global communicable disease-mediated mortality. Neutrophils play a leading role in trying to contain bacterial lung infection, but they also drive detrimental pulmonary inflammation, when dysregulated. Here we aimed at understanding the role of microRNA-223 in orchestrating pulmonary inflammation during pneumococcal pneumonia. Serum microRNA-223 was measured in patients with pneumococcal pneumonia and in healthy subjects. Pulmonary inflammation in wild-type and microRNA-223-knockout mice was assessed in terms of disease course, histopathology, cellular recruitment and evaluation of inflammatory protein and gene signatures following pneumococcal infection. Low levels of serum microRNA-223 correlated with increased disease severity in pneumococcal pneumonia patients. Prolonged neutrophilic influx into the lungs and alveolar spaces was detected in pneumococci-infected microRNA-223-knockout mice, possibly accounting for aggravated histopathology and acute lung injury. Expression of microRNA-223 in wild-type mice was induced by pneumococcal infection in a time-dependent manner in whole lungs and lung neutrophils. Single-cell transcriptome analyses of murine lungs revealed a unique profile of antimicrobial and cellular maturation genes that are dysregulated in neutrophils lacking microRNA-223. Taken together, low levels of microRNA-223 in human pneumonia patient serum were associated with increased disease severity, whilst its absence provoked dysregulation of the neutrophil transcriptome in murine pneumococcal pneumonia

Single-cell-resolved interspecies comparison shows a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19

A key issue for research on COVID-19 pathogenesis is the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leverage the model to molecularly survey the disease progression from time-resolved single-cell RNA sequencing data collected from healthy and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Syrian and Roborovski hamster lungs. We compare our data to human COVID-19 studies, including bronchoalveolar lavage, nasal swab, and postmortem lung tissue, and identify a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1- or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively

A pulmonologist's guide to perform and analyse cross-species single lung cell transcriptomics

Single-cell ribonucleic acid sequencing is becoming widely employed to study biological processes at a novel resolution depth. The ability to analyse transcriptomes of multiple heterogeneous cell types in parallel is especially valuable for cell-focused lung research where a variety of resident and recruited cells are essential for maintaining organ functionality. We compared the single-cell transcriptomes from publicly available and unpublished datasets of the lungs in six different species: human (Homo sapiens), African green monkey (Chlorocebus sabaeus), pig (Sus domesticus), hamster (Mesocricetus auratus), rat (Rattus norvegicus) and mouse (Mus musculus) by employing RNA velocity and intercellular communication based on ligand-receptor co-expression, among other techniques. Specifically, we demonstrated a workflow for interspecies data integration, applied a single unified gene nomenclature, performed cell-specific clustering and identified marker genes for each species. Overall, integrative approaches combining newly sequenced as well as publicly available datasets could help identify species-specific transcriptomic signatures in both healthy and diseased lung tissue and select appropriate models for future respiratory research

Key benefits of dexamethasone and antibody treatment in COVID-19 hamster models revealed by single cell transcriptomics

For COVID-19, effective and well-understood treatment options are still scarce. Since vaccine efficacy is challenged by novel variants, short-lasting immunity and vaccine hesitancy, understanding and optimizing therapeutic options remains essential. We aimed at better understanding the effects of two standard-of-care drugs, dexamethasone and anti-SARS-CoV-2 antibodies, on infection and host responses. By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of single or combinatorial treatments. Pulmonary viral burden was reduced by anti-SARS-CoV-2 antibody treatment, and similar or increased by dexamethasone alone. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment, and identified a specifically responsive subpopulation of neutrophils, thereby indicating a potential mechanism of action. Our analyses confirm the anti-inflammatory properties of dexamethasone and suggest possible mechanisms, validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and reveal synergistic effects of a combination therapy, thus informing more effective COVID-19 therapies