RESUMEN
Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-kB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138Aalteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-kB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1, which stimulated keratinocyte metabolism, but not for NF-kB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis.
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SNPs in the FAM13A locus are amongst the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases, however the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: 'long' and 'short', but their functions remain unknown, partly due to a lack of isoform conservation in mice. We performed in-depth characterisation of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate RhoGAP activity of this domain. In Xenopus laevis, which conserve the long isoform, Fam13a-deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long isoform deficiency did not affect multiciliogenesis but reduced cilia co-ordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform co-ordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.
Asunto(s)
Antiinflamatorios/uso terapéutico , Compuestos de Boro/uso terapéutico , Inflamación/tratamiento farmacológico , Calicreínas/antagonistas & inhibidores , Síndrome de Netherton/tratamiento farmacológico , Piel/patología , Administración Tópica , Animales , Modelos Animales de Enfermedad , Humanos , Calicreínas/genética , Ratones , Ratones Transgénicos , Transducción de Señal , Piel/efectos de los fármacos , Crema para la PielRESUMEN
To investigate how the CARD14E138A psoriasis-associated mutation induces skin inflammation, a knock-in mouse strain was generated that allows tamoxifen-induced expression of the homologous Card14E138A mutation from the endogenous mouse Card14 locus. Heterozygous expression of CARD14E138A rapidly induced skin acanthosis, immune cell infiltration and expression of psoriasis-associated pro-inflammatory genes. Homozygous expression of CARD14E138A induced more extensive skin inflammation and a severe systemic disease involving infiltration of myeloid cells in multiple organs, temperature reduction, weight loss and organ failure. This severe phenotype resembled acute exacerbations of generalised pustular psoriasis (GPP), a rare form of psoriasis that can be caused by CARD14 mutations in patients. CARD14E138A-induced skin inflammation and systemic disease were independent of adaptive immune cells, ameliorated by blocking TNF and induced by CARD14E138A signalling only in keratinocytes. These results suggest that anti-inflammatory therapies specifically targeting keratinocytes, rather than systemic biologicals, might be effective for GPP treatment early in disease progression.
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Proteínas Adaptadoras de Señalización CARD/genética , Dermatitis/genética , Guanilato-Quinasas/genética , Mutación , Psoriasis/genética , Síndrome de Respuesta Inflamatoria Sistémica/genética , Factor de Necrosis Tumoral alfa/genética , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Dermatitis/inmunología , Femenino , Guanilato-Quinasas/metabolismo , Masculino , Ratones , Psoriasis/inmunología , Síndrome de Respuesta Inflamatoria Sistémica/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Pattern recognition receptors detect microbial products and induce cytokines, which shape the immunological response. IL-12, TNF-α, and IL-1ß are proinflammatory cytokines, which are essential for resistance against infection, but when produced at high levels they may contribute to immunopathology. In contrast, IL-10 is an immunosuppressive cytokine, which dampens proinflammatory responses, but it can also lead to defective pathogen clearance. The regulation of these cytokines is therefore central to the generation of an effective but balanced immune response. In this study, we show that macrophages derived from C57BL/6 mice produce low levels of IL-12, TNF-α, and IL-1ß, but high levels of IL-10, in response to TLR4 and TLR2 ligands LPS and Pam3CSK4, as well as Burkholderia pseudomallei, a Gram-negative bacterium that activates TLR2/4. In contrast, macrophages derived from BALB/c mice show a reciprocal pattern of cytokine production. Differential production of IL-10 in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages was due to a type I IFN and ERK1/2-dependent, but IL-27-independent, mechanism. Enhanced type I IFN expression in LPS-stimulated C57BL/6 macrophages was accompanied by increased STAT1 and IFN regulatory factor 3 activation. Furthermore, type I IFN contributed to differential IL-1ß and IL-12 production in B. pseudomallei and LPS-stimulated C57BL/6 and BALB/c macrophages via both IL-10-dependent and -independent mechanisms. These findings highlight key pathways responsible for the regulation of pro- and anti-inflammatory cytokines in macrophages and reveal how they may differ according to the genetic background of the host.
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Citocinas/biosíntesis , Inflamación/inmunología , Interferón Tipo I/biosíntesis , Interleucina-10/análisis , Macrófagos/metabolismo , Animales , Burkholderia pseudomallei/inmunología , Citocinas/inmunología , Interferón Tipo I/inmunología , Interleucina-10/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Inherited and de novo mutations in the CARD14 gene promote the development of psoriasis, an inflammatory disease of the skin. Caspase recruitment domain-containing protein 14 (CARD14) is a member of the CARMA protein family that includes the structurally related CARD11 adaptor that mediates NF-κB activation by antigen receptors. We investigated the mechanism by which CARD14 mutation in psoriasis activates NF-κB. In contrast with wild-type CARD14, CARD14(E138A) and CARD14(G117S) psoriasis mutants interacted constitutively with BCL10 and MALT1, and triggered BCL10- and MALT1-dependent activation of NF-κB in keratinocytes. These alterations disrupted the inhibitory effect of the CARD14 linker region (LR) on NF-κB activation by facilitating BCL10 binding. Therefore, psoriasis mutations activated CARD14 by a mechanism analogous to oncogenic CARD11 mutations in non-Hodgkin B cell lymphomas. CARD14(E138A) also stimulated MALT1 paracaspase activity and activated both ERK1/2 and p38α MAP kinases. Inhibition of MALT1 with mepazine reduced CARD14(E138A)-induced expression of specific psoriasis-associated transcripts in keratinocytes. Our results establish the mechanism whereby gain-of-function CARD14 variants, which induce psoriatic disease in affected individuals, activate pro-inflammatory signalling.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasas/metabolismo , Guanilato Ciclasa/metabolismo , Proteínas de la Membrana/metabolismo , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Psoriasis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteína 10 de la LLC-Linfoma de Células B , Proteínas Adaptadoras de Señalización CARD/genética , Caspasas/genética , Línea Celular , Guanilato Ciclasa/genética , Humanos , Inmunoprecipitación , Queratinocitos/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas de la Membrana/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Mutación/genética , Proteínas de Neoplasias/genética , Unión Proteica/genética , Unión Proteica/fisiología , Psoriasis/genética , ARN Interferente Pequeño , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Analysis of the mouse transcriptional response to Listeria monocytogenes infection reveals that a large set of genes are perturbed in both blood and tissue and that these transcriptional responses are enriched for pathways of the immune response. Further we identified enrichment for both type I and type II interferon (IFN) signaling molecules in the blood and tissues upon infection. Since type I IFN signaling has been reported widely to impair bacterial clearance we examined gene expression from blood and tissues of wild type (WT) and type I IFNαß receptor-deficient (Ifnar1-/-) mice at the basal level and upon infection with L. monocytogenes. Measurement of the fold change response upon infection in the absence of type I IFN signaling demonstrated an upregulation of specific genes at day 1 post infection. A less marked reduction of the global gene expression signature in blood or tissues from infected Ifnar1-/- as compared to WT mice was observed at days 2 and 3 after infection, with marked reduction in key genes such as Oasg1 and Stat2. Moreover, on in depth analysis, changes in gene expression in uninfected mice of key IFN regulatory genes including Irf9, Irf7, Stat1 and others were identified, and although induced by an equivalent degree upon infection this resulted in significantly lower final gene expression levels upon infection of Ifnar1-/- mice. These data highlight how dysregulation of this network in the steady state and temporally upon infection may determine the outcome of this bacterial infection and how basal levels of type I IFN-inducible genes may perturb an optimal host immune response to control intracellular bacterial infections such as L. monocytogenes.
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Interferón Tipo I/fisiología , Listeriosis/inmunología , Transcripción Genética/inmunología , Transcriptoma , Animales , Células Sanguíneas/metabolismo , Resistencia a la Enfermedad , Regulación de la Expresión Génica/inmunología , Interferón gamma/fisiología , Listeriosis/genética , Listeriosis/metabolismo , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/deficiencia , Transducción de Señal , Bazo/metabolismo , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Tuberculosis, caused by the intracellular bacterium Mycobacterium tuberculosis, currently causes â¼1.4 million deaths per year, and it therefore remains a leading global health problem. The immune response during tuberculosis remains incompletely understood, particularly regarding immune factors that are harmful rather than protective to the host. Overproduction of the type I IFN family of cytokines is associated with exacerbated tuberculosis in both mouse models and in humans, although the mechanisms by which type I IFN promotes disease are not well understood. We have investigated the effect of type I IFN on M. tuberculosis-infected macrophages and found that production of host-protective cytokines such as TNF-α, IL-12, and IL-1ß is inhibited by exogenous type I IFN, whereas production of immunosuppressive IL-10 is promoted in an IL-27-independent manner. Furthermore, much of the ability of type I IFN to inhibit cytokine production was mediated by IL-10. Additionally, type I IFN compromised macrophage activation by the lymphoid immune response through severely disrupting responsiveness to IFN-γ, including M. tuberculosis killing. These findings describe important mechanisms by which type I IFN inhibits the immune response during tuberculosis.
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Interferón Tipo I/inmunología , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-12/inmunología , Interleucinas/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Interferón Tipo I/genética , Interferón gamma/genética , Interleucina-10/genética , Interleucina-12/genética , Interleucina-1beta/inmunología , Interleucinas/genética , Activación de Macrófagos/inmunología , Macrófagos/microbiología , Macrófagos/patología , Ratones , Ratones Noqueados , Tuberculosis/genética , Tuberculosis/patología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Interleukin (IL)-10 is an important immunoregulatory cytokine and an understanding of how IL-10 expression is controlled is critical in the design of immune intervention strategies. IL-10 is produced by almost all cell types within the innate (including macrophages, monocytes, dendritic cells (DCs), mast cells, neutrophils, eosinophils and natural killer cells) and adaptive (including CD4(+) T cells, CD8(+) T cells and B cells) immune systems. The mechanisms of IL-10 regulation operate at several stages including chromatin remodelling at the Il10 locus, transcriptional regulation of Il10 expression and post-transcriptional regulation of Il10 mRNA. In addition, whereas some aspects of Il10 gene regulation are conserved between different immune cell types, several are cell type- or stimulus-specific. Here, we outline the complexity of IL-10 production by discussing what is known about its regulation in macrophages, monocytes, DCs and CD4(+) T helper cells.
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Regulación de la Expresión Génica , Interleucina-10/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Mapeo Cromosómico , Células Dendríticas/inmunología , Humanos , Macrófagos/inmunologíaRESUMEN
The use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS), human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the early transcriptional kinetic response of innate cells to TLR ligands. Our study demonstrates that a transcriptomic analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.
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Sangre/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Humanos , Técnicas In Vitro , Lipopéptidos/metabolismo , Lipopolisacáridos/metabolismo , Análisis por Micromatrices , FN-kappa B/metabolismo , Factores de TiempoRESUMEN
The activation of TLRs by microbial molecules triggers intracellular-signaling cascades and the expression of cytokines such as IL-10. Il10 expression is tightly controlled to ensure effective immune responses, while preventing pathology. Maximal TLR-induction of Il10 transcription in macrophages requires signaling through the MAPKs, ERK, and p38. Signals via p38 downstream of TLR4 activation also regulate IL-10 at the post-transcriptional level, but whether this mechanism operates downstream of other TLRs is not clear. We compared the regulation of IL-10 production in TLR2 and TLR4-stimulated BM-derived macrophages and found different stability profiles for the Il10 mRNA. TLR2 signals promoted a rapid induction and degradation of Il10 mRNA, whereas TLR4 signals protected Il10 mRNA from rapid degradation, due to the activation of Toll/IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) and enhanced p38 signaling. This differential post-transcriptional mechanism contributes to a stronger induction of IL-10 secretion via TLR4. Our study provides a molecular mechanism for the differential IL-10 production by TLR2- or TLR4-stimulated BMMs, showing that p38-induced stability is not common to all TLR-signaling pathways. This mechanism is also observed upon bacterial activation of TLR2 or TLR4 in BMMs, contributing to IL-10 modulation in these cells in an infection setting.
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Interleucina-10/genética , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Procesamiento Postranscripcional del ARN , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Bacterias/inmunología , Activación Enzimática , Femenino , Regulación de la Expresión Génica , Macrófagos/microbiología , Ratones , Ratones Noqueados , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Tristetraprolina/deficiencia , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
As Nature Reviews Immunology reaches its 10(th) anniversary, the authors of one of the top-cited articles from each year take a trip down memory lane. We've asked them to look back on the state of research at the time their Review was published, to consider why the article has had the impact it has and to discuss the future directions of their field. This Viewpoint article provides an interesting snapshot of some of the fundamental advances in immunology over the past 10 years. Highlights include our improved understanding of Toll-like receptor signalling, and of immune regulation mediated by regulatory T cells, indoleamine 2,3-dioxygenase, myeloid-derived suppressor cells and interleukin-10. The complexities in the development and heterogeneity of macrophages, dendritic cells and T helper cells continue to engage immunologists, as do the immune processes involved in diseases such as atherosclerosis. We look forward to what the next 10 years of immunology research may bring.