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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.
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Dermatite Atópica , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Queratinócitos/metabolismo , Epiderme/metabolismo , Inflamação , Diferenciação Celular , Infecções Estafilocócicas/patologiaRESUMO
Langerhans cells (LCs), residing in the epidermis, are able to induce potent immunogenic responses and also to mediate immune tolerance. We propose that tolerogenic and immunogenic responses of LCs are directed by signaling from the epidermis and involve counter-acting gene circuits that are coupled to a core maturation gene module. We base our analysis on recent genetic and genomic findings facilitating the understanding of the molecular mechanisms controlling these divergent immune functions. Comparing gene regulatory network (GRN) analyses of various types of dendritic cells (DCs) including LCs we integrate signaling-dependent (TGFß, EpCAM, ß-Catenin) and transcription factor (IRF4, IRF1, NFκB) regulated gene circuits that appear to orchestrate the distinctive LC functional states. Our model proposes, that while epidermal signaling in the steady-state promotes LC tolerogenic function, the disruption of cell-cell contacts coupled with inflammatory signaling induces LC immunogenic programing. The conceptual framework emphasizes the sensing of discrete epidermal and inflammatory cues by resident LCs in dictating their genomic programing and cell state dynamics.
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Redes Reguladoras de Genes , Células de Langerhans , Epiderme/metabolismo , Células de Langerhans/metabolismo , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4+ Th cells. During severe viral infection and chronic inflammation, the submucosa is susceptible to bacterial invasion by lung microbiota such as nontypeable Haemophilus influenzae (NTHi). Given their proximity in tissue, we hypothesized that human lung fibroblasts play an important role in modulating Th cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4+ T cell Ag-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to IFN-γ. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. Although unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the costimulatory molecules 4-1BBL, OX-40L, and CD70, all of which are related to memory T cell activation and maintenance. CD4+ T cells isolated from the lung were predominantly (mean 97.5%) CD45RO+ memory cells. Finally, cultured fibroblasts activated IFN-γ and IL-17A cytokine production by autologous, NTHi-specific lung CD4+ T cells, and cytokine production was inhibited by a HLA-DR blocking Ab. These results indicate a novel role for human lung fibroblasts in contributing to responses against bacterial infection through activation of bacteria-specific CD4+ T cells.
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Apresentação de Antígeno/imunologia , Fibroblastos/imunologia , Pulmão/imunologia , Ativação Linfocitária/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos de Bactérias/imunologia , Células Cultivadas , Técnicas de Cocultura , Citometria de Fluxo , Infecções por Haemophilus/imunologia , Haemophilus influenzae/imunologia , Humanos , Hibridização in Situ FluorescenteRESUMO
BACKGROUND: Previous reports have demonstrated the utility of T-cell proliferation and cytokine release assays as in vitro diagnostic tests for drug causation in drug hypersensitivity reactions (DHR). However, data from pediatric populations are scarce compared with data in adults. OBJECTIVE: To compare the lymphocyte proliferation assay (LPA) with combination cytokine assays in the pediatric population and to identify its potential use in the acute and postrecovery phases. METHODS: A total of 18 in vitro tests were undertaken ex vivo to compare drug-specific proliferation and cytokine release (interferon-γ [IFN-γ] and interleukin-4 [IL-4]). The study included 16 patients with DHR: 7 children tested in the acute phase, 7 tested after recovery, and 2 tested during both the acute and postrecovery phases. RESULTS: The sensitivity of the LPA was better during the acute stage of DHR in children. Cytokine assays revealed a higher frequency of positive drug-specific responses compared with LPA in both the acute (LPA, 77.8%; IFN-γ, 88.9%; IL-4, 100%) and postrecovery phases (LPA, 33.3%; IFN-γ, 66.7%; IL-4, 66.7%). Combination cytokine assays (IFN-γ and IL-4) produced higher positive drug-specific responses in identifying culprit drugs compared with LPA in both the acute and postrecovery phases. CONCLUSION: In vitro drug-induced T-cell proliferation and cytokine release assays are useful for identification of the causative drug in children with DHR. Cytokine assays (IFN-γ and IL-4) were better than LPA, but when combined, they offer even greater utility in the diagnosis of acute and postrecovery DHR. Cytokine detection is rapid and does not involve radioactivity. These novel in vitro assays may offer a significant advancement in our future management of DHR in children.
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Testes Diagnósticos de Rotina , Hipersensibilidade a Drogas/diagnóstico , Hipersensibilidade a Drogas/imunologia , Adolescente , Criança , Pré-Escolar , Citocinas/metabolismo , Testes Diagnósticos de Rotina/métodos , Hipersensibilidade a Drogas/metabolismo , Feminino , Humanos , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Fenótipo , Índice de Gravidade de DoençaRESUMO
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.
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Dermatite Atópica , Animais , Humanos , Dermatite Atópica/genética , Alérgenos , Inflamação/genética , Pele , PyroglyphidaeRESUMO
Mycobacterium tuberculosis (Mtb) causes the human disease tuberculosis (TB) and remains the top global infectious pandemic after coronavirus disease 2019 (COVID-19). Furthermore, TB has killed many more humans than any other pathogen, after prolonged coevolution to optimise its pathogenic strategies. Full understanding of fundamental disease processes in humans is necessary to successfully combat this highly successful pathogen. While the importance of immunodeficiency has been long recognised, biologic therapies and unbiased approaches are providing unprecedented insights into the intricacy of the host-pathogen interaction. The nature of a protective response is more complex than previously hypothesised. Here, we integrate recent evidence from human studies and unbiased approaches to consider how Mtb causes human TB and highlight the recurring theme of extracellular matrix (ECM) turnover.
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COVID-19 , Mycobacterium tuberculosis , Tuberculose , Granuloma , Interações Hospedeiro-Patógeno , Humanos , SARS-CoV-2RESUMO
Human epidermal Langerhans cells (LCs) maintain immune homeostasis in the skin. To examine transcriptional programming of human primary LCs during homeostasis, we performed scRNA-seq analysis of LCs before and after migration from the epidermis, coupled with functional assessment of their regulatory T cell priming capabilities. The analysis revealed that steady-state LCs exist in a continuum of maturation states and upregulate antigen presentation genes along with an immunoregulatory module including the genes IDO1, LGALS1, LAMTOR1, IL4I, upon their migration. The migration-induced transition in genomic state is accompanied by the ability of LCs to more efficiently prime regulatory T cell responses in co-culture assays. Computational analyses of the scRNAseq datasets using SCENIC and Partial Information Decomposition in Context identified a set of migration-induced transcription factors including IRF4, KLF6 and RelB as key nodes within a immunoregulatory gene regulatory network. These findings support a model in which efficient priming of immunoregulatory responses by LCs is dependent on coordinated upregulation of a migration-coupled maturation program with a immunoregulation-promoting genomic module.
Assuntos
Galectina 1 , Células de Langerhans , Movimento Celular/genética , Epiderme , Humanos , PeleRESUMO
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 (ß = -0.702, P = 4.3 × 10-5) and increased NFKBIA (ß = 0.486, P = .001), reflecting reduced NFKB signal activation. Deleterious variation in the NOD2-RIPK2 complex was associated with increased NLRP3 (ß = 0.8, P = 3.1475 × 10-8) and TXN (ß = -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 (ß = -0.677, P = 1.7 × 10-5), a key signal transduction protein in the NOD2 signaling cascade and increased IFNA1 (ß = 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.
Assuntos
Doenças Inflamatórias Intestinais , Proteína Adaptadora de Sinalização NOD2 , Criança , Variação Genética , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais/genética , Regulação para CimaRESUMO
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.
Assuntos
Leishmania major , Leishmania mexicana , Leishmaniose , Citocinas/metabolismo , Redes Reguladoras de Genes , Humanos , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , MacrófagosRESUMO
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-1ß 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.
Assuntos
Doença Enxerto-Hospedeiro , Pele , Animais , Humanos , Tolerância Imunológica , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , Pele/metabolismo , Linfócitos T ReguladoresRESUMO
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 0·0 (IQR 0·0-0·0) versus 5·0 (1·5-10·5) for IgA-secreting plasma cells (p<0·0001), and 0·0 (0·0-0·0) versus 3·0 (1·5-9·5) for IgG-secreting plasma cells (p<0·0001). Median N lactamica-specific IgG memory-B-cell frequencies (percentage of total IgG memory B cells) increased from 0·0024% (0·0000-0·0097) at baseline to 0·0384% (0·0275-0·0649) at day 28 (p<0·0001). The frequency of N meningitidis-specific IgA-secreting and IgG-secreting 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>0·99). 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.
Assuntos
Neisseria lactamica , Neisseria meningitidis , Adulto , Humanos , Leucócitos Mononucleares , Imunoglobulina A Secretora , Fosfatos , Solução Salina , Imunoglobulina GRESUMO
Background: The COVID-19 pandemic has created pressure on healthcare systems worldwide. Tools that can stratify individuals according to prognosis could allow for more efficient allocation of healthcare resources and thus improved patient outcomes. It is currently unclear if blood gene expression signatures derived from patients at the point of admission to hospital could provide useful prognostic information. Methods: Gene expression of whole blood obtained at the point of admission from a cohort of 78 patients hospitalised with COVID-19 during the first wave was measured by high resolution RNA sequencing. Gene signatures predictive of admission to Intensive Care Unit were identified and tested using machine learning and topological data analysis, TopMD. Results: The best gene expression signature predictive of ICU admission was defined using topological data analysis with an accuracy: 0.72 and ROC AUC: 0.76. The gene signature was primarily based on differentially activated pathways controlling epidermal growth factor receptor (EGFR) presentation, Peroxisome proliferator-activated receptor alpha (PPAR-α) signalling and Transforming growth factor beta (TGF-ß) signalling. Conclusions: Gene expression signatures from blood taken at the point of admission to hospital predicted ICU admission of treatment naïve patients with COVID-19.
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COVID-19 , COVID-19/genética , Receptores ErbB , Expressão Gênica , Humanos , Unidades de Terapia Intensiva , PPAR alfa , Pandemias , Fator de Crescimento Transformador betaRESUMO
The worldwide COVID-19 pandemic has claimed millions of lives and has had a profound effect on global life. Understanding the body's immune response to SARS-CoV-2 infection is crucial in improving patient management and prognosis. In this study we compared influenza and SARS-CoV-2 infected patient cohorts to identify distinct blood transcript abundances and cellular composition to better understand the natural immune response associated with COVID-19, compared to another viral infection being influenza, and identify a prognostic signature of COVID-19 patient outcome. Clinical characteristics and peripheral blood were acquired upon hospital admission from two well characterised cohorts, a cohort of 88 patients infected with influenza and a cohort of 80 patients infected with SARS-CoV-2 during the first wave of the pandemic and prior to availability of COVID-19 treatments and vaccines. Gene transcript abundances, enriched pathways and cellular composition were compared between cohorts using RNA-seq. A genetic signature between COVID-19 survivors and non-survivors was assessed as a prognostic predictor of COVID-19 outcome. Contrasting immune responses were detected with an innate response elevated in influenza and an adaptive response elevated in COVID-19. Additionally ribosomal, mitochondrial oxidative stress and interferon signalling pathways differentiated the cohorts. An adaptive immune response was associated with COVID-19 survival, while an inflammatory response predicted death. A prognostic transcript signature, associated with circulating immunoglobulins, nucleosome assembly, cytokine production and T cell activation, was able to stratify COVID-19 patients likely to survive or die. This study provides a unique insight into the immune responses of treatment naïve patients with influenza or COVID-19. The comparison of immune response between COVID-19 survivors and non-survivors enables prognostication of COVID-19 patients and may suggest potential therapeutic strategies to improve survival.
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COVID-19 , Vacinas contra Influenza , Influenza Humana , Imunidade Adaptativa , Humanos , Pandemias , SARS-CoV-2RESUMO
Single-cell transcriptomics suffer from sensitivity limits that restrict low abundance transcript identification, affects clustering and can hamper downstream analyses. Here, we describe Constellation sequencing (Constellation-Seq), a molecular transcriptome filter that delivers two orders of magnitude sensitivity gains by maximizing read utility while reducing the data sparsity and sequencing costs. The technique reliably measures changes in gene expression and was demonstrated by resolving rare dendritic cell populations from a peripheral blood mononuclear cell sample sample and exploring their biology with extreme resolution. The simple and powerful method is fully compatible with standard scRNA-Seq library preparation protocols and can be used for hypothesis testing, marker validation or investigating pathways.
RESUMO
BACKGROUND: Natural killer (NK) cells are increasingly being recognized as agents for cancer immunotherapy. The killer cell immunoglobulin-like receptors (KIRs) are expressed by NK cells and are immunogenetic determinants of the outcome of cancer. In particular, KIR2DS2 is associated with protective responses to several cancers and also direct recognition of cancer targets in vitro. Due to the high homology between activating and inhibitory KIR genes to date, it has been challenging to target individual KIR for therapeutic benefit. METHODS: A novel KIR2DS2-targeting therapeutic peptide:MHC DNA vaccine was designed and used to immunize mice transgenic for KIR genes (KIR-Tg). NK cells were isolated from the livers and spleens of vaccinated mice and then analyzed for activation by flow cytometry, RNA profiling and cytotoxicity assays. In vivo assays of NK cell function using a syngeneic cancer model (B16 melanoma) and an adoptive transfer model for human hepatocellular carcinoma (Huh7) were performed. RESULTS: Injecting KIR-Tg mice with the vaccine construct activated NK cells in both liver and spleens of mice, with preferential activation of KIR2DS2-positive NK cells. KIR-specific activation was most marked on the CD11b+CD27+ mature subset of NK cells. RNA profiling indicated that the DNA vaccine upregulated genes associated with cellular metabolism and downregulated genes related to histone H3 methylation, which are associated with immune cell maturation and NK cell function. Vaccination led to canonical and cross-reactive peptide:MHC-specific NK cell responses. In vivo, DNA vaccination led to enhanced antitumor responses against B16F10 melanoma cells and also enhanced responses against a tumor model expressing the KIR2DS2 ligand HLA-C*0102. CONCLUSION: We show the feasibility of a peptide-based KIR-targeting vaccine strategy to activate NK cells and hence generate functional antitumor responses. This approach does not require detailed knowledge of the tumor peptidomes nor HLA matching with the patient. It therefore offers a novel opportunity for targeting NK cells for cancer immunotherapy.
Assuntos
Vacinas Anticâncer/administração & dosagem , Citotoxicidade Imunológica/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Receptores KIR/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Vacinas de DNA/administração & dosagem , Animais , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Antígenos HLA-C/administração & dosagem , Antígenos HLA-C/genética , Antígenos HLA-C/imunologia , Haplótipos , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/administração & dosagem , Peptídeos/genética , Peptídeos/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Receptores KIR/genética , Receptores KIR/imunologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Vacinação , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
Langerhans cells (LCs) reside in the epidermis as a dense network of immune system sentinels, coordinating both immunogenic and tolerogenic immune responses. To determine molecular switches directing induction of LC immune activation, we performed mathematical modelling of gene regulatory networks identified by single cell RNA sequencing of LCs exposed to TNF-alpha, a key pro-inflammatory signal produced by the skin. Our approach delineated three programmes of LC phenotypic activation (immunogenic, tolerogenic or ambivalent), and confirmed that TNF-alpha enhanced LC immunogenic programming. Through regulon analysis followed by mutual information modelling, we identified IRF1 as the key transcription factor for the regulation of immunogenicity in LCs. Application of a mathematical toggle switch model, coupling IRF1 with tolerance-inducing transcription factors, determined the key set of transcription factors regulating the switch between tolerance and immunogenicity, and correctly predicted LC behaviour in LCs derived from different body sites. Our findings provide a mechanistic explanation of how combinatorial interactions between different transcription factors can coordinate specific transcriptional programmes in human LCs, interpreting the microenvironmental context of the local tissue microenvironments.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Células de Langerhans/imunologia , Células de Langerhans/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Epiderme/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Fatores Reguladores de Interferon/genética , Transdução de Sinais , Transcrição Gênica , Fator de Necrose Tumoral alfa/genéticaRESUMO
The future of single cell diversity screens involves ever-larger sample sizes, dictating the need for higher throughput methods with low analytical noise to accurately describe the nature of the cellular system. Current approaches are limited by the Poisson statistic, requiring dilute cell suspensions and associated losses in throughput. In this contribution, we apply Dean entrainment to both cell and bead inputs, defining different volume packets to effect efficient co-encapsulation. Volume ratio scaling was explored to identify optimal conditions. This enabled the co-encapsulation of single cells with reporter beads at rates of â¼1 million cells per hour, while increasing assay signal-to-noise with cell multiplet rates of â¼2.5% and capturing â¼70% of cells. The method, called Pirouette coupling, extends our capacity to investigate biological systems.
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Bioensaio , Análise de Célula Única , RuídoRESUMO
Tuberculosis (TB) is a persistent global pandemic, and standard treatment for it has not changed for 30 years. Mycobacterium tuberculosis (Mtb) has undergone prolonged coevolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesized that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a noninfectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared 3 primary human cell culture models at the whole transcriptome level and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified 12 intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.
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Granuloma do Sistema Respiratório/metabolismo , Pulmão/metabolismo , Modelos Biológicos , Mycobacterium tuberculosis/metabolismo , RNA-Seq , Tuberculose Pulmonar/metabolismo , Adulto , Idoso , Feminino , Granuloma do Sistema Respiratório/genética , Granuloma do Sistema Respiratório/microbiologia , Granuloma do Sistema Respiratório/patologia , Humanos , Pulmão/microbiologia , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/patologiaRESUMO
BACKGROUND AND AIMS: Crohn's disease [CD] arises through host-environment interaction. Abnormal gene expression results from disturbed pathway activation or response to bacteria. We aimed to determine activated pathways and driving cell types in paediatric CD. METHODS: We employed contemporary targeted autoimmune RNA sequencing, in parallel to single-cell sequencing, to ileal tissue derived from paediatric CD and controls. Weighted gene co-expression network analysis [WGCNA] was performed and differentially expressed genes [DEGs] were determined. We integrated clinical data to determine co-expression modules associated with outcomes. RESULTS: In all, 27 treatment-naive CD [TN-CD], 26 established CD patients and 17 controls were included. WGCNA revealed a 31-gene signature characterising TN-CD patients, but not established CD, nor controls. The CSF3R gene is a hub within this module and is key in neutrophil expansion and differentiation. Antimicrobial genes, including S100A12 and the calprotectin subunit S100A9, were significantly upregulated in TN CD compared with controls [p = 2.61 x 10-15 and p = 9.13 x 10-14, respectively] and established CD [both p = 0.0055]. Gene-enrichment analysis confirmed upregulation of the IL17-, NOD- and Oncostatin-M-signalling pathways in TN-CD patients, identified in both WGCNA and DEG analyses. An upregulated gene signature was enriched for transcripts promoting Th17-cell differentiation and correlated with prolonged time to relapse [correlation-coefficient-0.36, p = 0.07]. Single-cell sequencing of TN-CD patients identified specialised epithelial cells driving differential expression of S100A9. Cell groups, determined by single-cell gene expression, demonstrated enrichment of IL17-signalling in monocytes and epithelial cells. CONCLUSIONS: Ileal tissue from treatment-naïve paediatric patients is significantly upregulated for genes driving IL17-, NOD- and Oncostatin-M-signalling. This signal is driven by a distinct subset of epithelial cells expressing antimicrobial gene transcripts.