Langerhans cells (LCs) are distinct among phagocytes, functioning both as embryo-derived, tissue-resident macrophages in skin innervation and repair and as migrating professional antigen-presenting cells, a function classically assigned to dendritic cells (DCs). Here, we demonstrate that both intrinsic and extrinsic factors imprint this dual identity. Using ablation of embryo-derived LCs in the murine adult skin and tracking differentiation of incoming monocyte-derived replacements, we found intrinsic intraepidermal heterogeneity. We observed that ontogenically distinct monocytes give rise to LCs. Within the epidermis, Jagged-dependent activation of Notch signaling, likely within the hair follicle niche, provided an initial site of LC commitment before metabolic adaptation and survival of monocyte-derived LCs. In the human skin, embryo-derived LCs in newborns retained transcriptional evidence of their macrophage origin, but this was superseded by DC-like immune modules after postnatal expansion. Thus, adaptation to adult skin niches replicates conditioning of LC at birth, permitting repair of the embryo-derived LC network.

Supplementary figure 5: Tumoral Treg suppression of anti-CD3 induced tumoral effector T cell proliferation can be inhibited by anti-OX40.

Regulation of cutaneous immunity is severely compromised in inflammatory skin disease. To investigate the molecular crosstalk underpinning tolerance versus inflammation in atopic dermatitis, we utilise a human in vivo allergen challenge study, exposing atopic dermatitis patients to house dust mite. Here we analyse transcriptional programmes at the population and single cell levels in parallel with immunophenotyping of cutaneous immunocytes revealed a distinct dichotomy in atopic dermatitis patient responsiveness to house dust mite challenge. Our study shows that reactivity to house dust mite was associated with high basal levels of TNF-expressing cutaneous Th17 T cells, and documents the presence of hub structures where Langerhans cells and T cells co-localised. Mechanistically, we identify expression of metallothioneins and transcriptional programmes encoding antioxidant defences across all skin cell types, that appear to protect against allergen-induced inflammation. Furthermore, single nucleotide polymorphisms in the MTIX gene are associated with patients who did not react to house dust mite, opening up possibilities for therapeutic interventions modulating metallothionein expression in atopic dermatitis.

Supplementary figure 2: CLA is expressed by tumoral T cells and Tregs in cSCCs.

Supplementary figure 6: Comparison of FOXP3+ Treg frequencies between well differentiated cSCCs (G1) and moderately or poorly differentiated cSCCs (G2-3).

Supplementary figure 4: cSCC blood vessels express e-selectin.

Supplementary figure 1: Characterization of the cSCC immune infiltrate demonstrates the presence of tumoral Tregs.

Background Acute cutaneous inflammation causes microbiome alterations as well as ultrastructural changes in epidermis stratification. However, the interactions between keratinocyte proliferation and differentiation status and the skin microbiome have not been fully explored. Objectives Hypothesizing that the skin microbiome contributes to regulation of keratinocyte differentiation and can modify antimicrobial responses, we examined the effect of exposure to commensal (Staphylococcus epidermidis, SE) or pathogenic (Staphylococcus aureus, SA) challenge on epidermal models. Methods Explant biopsies were taken to investigate species-specific antimicrobial effects of host factors. Further investigations were performed in reconstituted epidermal models by bulk transcriptomic analysis alongside secreted protein profiling. Single-cell RNA sequencing analysis was performed to explore the keratinocyte populations responsible for SA inflammation. A dataset of 6391 keratinocytes from control (2044 cells), SE challenge (2028 cells) and SA challenge (2319 cells) was generated from reconstituted epidermal models. Results Bacterial lawns of SA, not SE, were inhibited by human skin explant samples, and microarray analysis of three-dimensional epidermis models showed that host antimicrobial peptide expression was induced by SE but not SA. Protein analysis of bacterial cocultured models showed that SA exposure induced inflammatory mediator expression, indicating keratinocyte activation of other epidermal immune populations. Single-cell DropSeq analysis of unchallenged naive, SE-challenged and SA-challenged epidermis models was undertaken to distinguish cells from basal, spinous and granular layers, and to interrogate them in relation to model exposure. In contrast to SE, SA specifically induced a subpopulation of spinous cells that highly expressed transcripts related to epidermal inflammation and antimicrobial response. Furthermore, SA, but not SE, specifically induced a basal population that highly expressed interleukin-1 alarmins. Conclusions These findings suggest that SA-associated remodelling of the epidermis is compartmentalized to different keratinocyte populations. Elucidating the mechanisms regulating bacterial sensing-triggered inflammatory responses within tissues will enable further understanding of microbiome dysbiosis and inflammatory skin diseases, such as atopic eczema.

Supplementary figure 3: Blood vessels are located in the peritumoral areas of cSCCs.

T cell pathology in the skin leads to monocyte influx, but we have little understanding of the fate of recruited cells within the diseased niche, or the long-term impact on cutaneous immune homeostasis. By combining a murine model of acute graft-versus-host disease (aGVHD) with analysis of patient samples, we demonstrate that pathology initiates dermis-specific macrophage differentiation and show that aGVHD-primed macrophages continue to dominate the dermal compartment at the relative expense of quiescent MHCIIint cells. Exposure of the altered dermal niche to topical haptens after disease resolution results in hyper-activation of regulatory T cells (Treg), but local breakdown in tolerance. Disease-imprinted macrophages express increased IL-1β and are predicted to elicit altered TNF superfamily interactions with cutaneous Treg, and we demonstrate the direct loss of T cell regulation within the resolved skin. Thus, T cell pathology leaves an immunological scar in the skin marked by failure to re-set immune homeostasis.

Leishmania amazonensis and Leishmania major are the causative agents of cutaneous and mucocutaneous diseases. The infections‘ outcome depends on host–parasite interactions and Th1/Th2 response, and in cutaneous form, regulation of Th17 cytokines has been reported to maintain inflammation in lesions. Despite that, the Th17 regulatory scenario remains unclear. With the aim to gain a better understanding of the transcription factors (TFs) and genes involved in Th17 induction, in this study, the role of inducing factors of the Th17 pathway in Leishmania–macrophage infection was addressed through computational modeling of gene regulatory networks (GRNs). The Th17 GRN modeling integrated experimentally validated data available in the literature and gene expression data from a time-series RNA-seq experiment (4, 24, 48, and 72 h post-infection). The generated model comprises a total of 10 TFs, 22 coding genes, and 16 cytokines related to the Th17 immune modulation. Addressing the Th17 induction in infected and uninfected macrophages, an increase of 2- to 3-fold in 4–24 h was observed in the former. However, there was a decrease in basal levels at 48–72 h for both groups. In order to evaluate the possible outcomes triggered by GRN component modulation in the Th17 pathway. The generated GRN models promoted an integrative and dynamic view of Leishmania–macrophage interaction over time that extends beyond the analysis of single-gene expression.

Abstract Background Inflammatory bowel disease may arise with inadequate immune response to intestinal bacteria. NOD2 is an established gene in Crohn’s disease pathogenesis, with deleterious variation associated with reduced NFKB signaling. We hypothesized that deleterious variation across the NOD2 signaling pathway impacts on transcription. Methods Treatment-naïve pediatric inflammatory bowel disease patients had ileal biopsies for targeted autoimmune RNA-sequencing and blood for whole exome sequencing collected at diagnostic endoscopy. Utilizing GenePy, a per-individual, per-gene score, genes within the NOD signaling pathway were assigned a quantitative score representing total variant burden. Where multiple genes formed complexes, GenePy scores were summed to create a “complex” score. Normalized transcript expression of 95 genes within this pathway was retrieved. Regression analysis was performed to determine the impact of genomic variation on gene transcription. Results Thirty-nine patients were included. Limited clustering of patients based on NOD signaling transcripts was related to underlying genomic variation. Patients harboring deleterious variation in NOD2 had reduced NOD2 (β = -0.702, P = 4.3 × 10-5) and increased NFKBIA (β = 0.486, P = .001), reflecting reduced NFKB signal activation. Deleterious variation in the NOD2-RIPK2 complex was associated with increased NLRP3 (β = 0.8, P = 3.1475 × 10-8) and TXN (β = -0.417, P = 8.4 × 10-5) transcription, components of the NLRP3 inflammasome. Deleterious variation in the TAK1-TAB complex resulted in reduced MAPK14 transcription (β = -0.677, P = 1.7 × 10-5), a key signal transduction protein in the NOD2 signaling cascade and increased IFNA1 (β = 0.479, P = .001), indicating reduced transcription of NFKB activators and alternative interferon transcription in these patients. Conclusions Data integration identified perturbation of NOD2 signaling transcription correlated with genomic variation. A hypoimmune NFKB signaling transcription response was observed. Alternative inflammatory pathways were activated and may represent therapeutic targets in specific patients.

Background Pharyngeal colonisation by the commensal bacterium Neisseria lactamica inhibits colonisation by Neisseria meningitidis and has an inverse epidemiological association with meningococcal disease. The mechanisms that underpin this relationship are unclear, but could involve the induction of cross-reactive immunity. In this study, we aimed to evaluate whether colonisation with N lactamica induces N lactamica-specific B-cell responses that are cross-reactive with N meningitidis. Methods In this randomised, placebo-controlled, human infection trial at University Hospital Southampton Clinical Research Facility (Southampton, UK), healthy adults aged 18-45 years were randomly assigned (2:1) to receive intranasal inoculation with either 105 colony-forming units of N lactamica in 1 mL phosphate-buffered saline (PBS) or 1 mL PBS alone. Participants and researchers conducting participant sampling and immunological assays were masked to allocation. The primary endpoint was the frequency of circulating N lactamica-specific plasma cells and memory B cells after N lactamica inoculation (day 7-28) compared with baseline values (day 0), measured using enzyme-linked immunospot assays. The secondary endpoint was to measure the frequency of N meningitidis-specific B cells. In a second study, we measured the effect of duration of N lactamica colonisation on seroconversion by terminating carriage at either 4 days or 14 days with single-dose oral ciprofloxacin. The studies are now closed to participants. The trials are registered with ClinicalTrials.gov, NCT03633474 and NCT03549325. Findings Of 50 participants assessed for eligibility between Sept 5, 2018, and March 3, 2019, 31 were randomly assigned (n=20 N lactamica, n=11 PBS). Among the 17 participants who were colonised with N lactamica, the median baselines compared with peak post-colonisation N lactamica-specific plasma-cell frequencies (per 105 peripheral blood mononuclear cells) were 0 center dot 0 (IQR 0 center dot 0-0 center dot 0) versus 5 center dot 0 (1 center dot 5-10 center dot 5) for IgA-secreting plasma cells (p<0 center dot 0001), and 0 center dot 0 (0 center dot 0-0 center dot 0) versus 3 center dot 0 (1 center dot 5-9 center dot 5) for IgG-secreting plasma cells (p<0 center dot 0001). Median N lactamica-specific IgG memoryB-cell frequencies (percentage of total IgG memory B cells) increased from 0 center dot 0024% (0 center dot 0000-0 center dot 0097) at baseline to 0 center dot 0384% (0 center dot 0275-0 center dot 0649) at day 28 (p<0 center dot 0001). The frequency of N meningitidis-specific IgA-secreting and IgGsecreting plasma cells and memory B cells also increased signficantly in participants who were colonised with N lactamica. Upper respiratory tract symptoms were reported in ten (50%) of 20 participants who were inoculated with N lactamica and six (55%) of 11 participants who were inoculated with PBS (p>0 center dot 99). Three additional adverse events (two in the N lactamica group and one in the PBS group) and no serious adverse events were reported. In the second study, anti-N lactamica and anti-N meningitidis serum IgG titres increased only in participants who were colonised with N lactamica for 14 days. Interpretation Natural immunity to N meningitidis after colonisation with N lactamica might be due to cross-reactive adaptive responses. Exploitation of this microbial mechanism with a genetically modified live vector could protect against N meningitidis colonisation and disease. Funding Wellcome Trust, Medical Research Council, and NIHR Southampton Biomedical Research Centre. Copyright (c) 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.

Respiratory diseases account for over 5 million deaths yearly and are a huge burden to healthcare systems worldwide. Murine models have been of paramount importance to decode human lung biology in vivo, but their genetic, anatomical, physiological and immunological differences with humans significantly hamper successful translation of research into clinical practice. Thus, to clearly understand human lung physiology, development, homeostasis and mechanistic dysregulation that may lead to disease, it is essential to develop models that accurately recreate the extraordinary complexity of the human pulmonary architecture and biology. Recent advances in micro-engineering technology and tissue engineering have allowed the development of more sophisticated models intending to bridge the gap between the native lung and its replicates in vitro Alongside advanced culture techniques, remarkable technological growth in downstream analyses has significantly increased the predictive power of human biology-based in vitro models by allowing capture and quantification of complex signals. Refined integrated multi-omics readouts could lead to an acceleration of the translational pipeline from in vitro experimental settings to drug development and clinical testing in the future. This review highlights the range and complexity of state-of-the-art lung models for different areas of the respiratory system, from nasal to large airways, small airways and alveoli, with consideration of various aspects of disease states and their potential applications, including pre-clinical drug testing. We explore how development of optimised physiologically relevant in vitro human lung models could accelerate the identification of novel therapeutics with increased potential to translate successfully from the bench to the patient's bedside.

The ability to reliably predict and infer cellular responses to environmental exposures would offer a major advance in the investigation of immune regulation in health and disease. One possible approach is the use of in silico modelling. Design of such a mathematical kinetic model would be based on existing knowledge of a biological system and utilise a partial data set to parameterise. However, the process of parameter estimation, key for the accuracy of the model, is difficult to conduct by hand, and thus a computational alternative is necessary. We report the utility of Genetic Algorithm with Rank Selection (GARS) as a parameter estimation tool on multiple biological models, including heat shock, signal transduction via ERK, circadian rhythm and NFκB systems, where it showed strong accuracy and superiority to the Extended Kalman Filter method, Algebraic Difference Equations, and MATLAB fminsearch approaches. GARS parameter estimation is a valuable tool for biological data because it reliably infers system behaviour from partial data sets, allowing for the prediction of cellular responses to environmental exposures.