The aryl hydrocarbon receptor (AhR) has been attributed with anti-inflammatory effects in the development of pathological immune responses leading to experimental autoimmune encephalomyelitis (EAE) via the induction of regulatory T cells. In agreement with previously published findings, we find that TCDD administration confers protection from EAE, however, this immuno-modulatory effect was not the consequence of de novo Treg generation, but the inhibition of Th17 cell differentiation. Systemic application of FICZ at the time of immunization also reduced EAE pathology albeit to a lesser degree than TCDD. In vitro Th17 differentiation in the presence of AhR agonists, including TCDD, promoted IL-17 and IL-22 expression, but did not induce Treg differentiation. AhR affinity influenced the amounts of IL-17 and IL-22 protein that was secreted by Th17 cells, but did not seem to affect susceptibility to EAE in vivo. Making use of conditional AhR-deficient mice, we show that the anti-inflammatory effect of TCDD depends on AhR activation in both T cells and dendritic cells, further emphasising the ability of TCDD to interfere with T effector cell differentiation in vivo. The dichotomy between the in vivo and in vitro effects of AhR reveals the complexity of the AhR pathway, which has the capacity of affecting different AhR-expressing cell types involved in mounting immune responses, thus participating in defining their outcome.
Sequence-based variation in gene expression is a key driver of disease risk. Common variants regulating expression in cis have been mapped in many eQTL studies typically in single tissues from unrelated individuals. Here, we present a comprehensive analysis of gene expression across multiple tissues conducted in a large set of mono- and dizygotic twins that allows systematic dissection of genetic (cis and trans) and non-genetic effects on gene expression. Using identity-by-descent estimates, we show that at least 40% of the total heritable cis-effect on expression cannot be accounted for by common cis-variants, a finding which exposes the contribution of low frequency and rare regulatory variants with respect to both transcriptional regulation and complex trait susceptibility. We show that a substantial proportion of gene expression heritability is trans to the structural gene and identify several replicating trans-variants which act predominantly in a tissue-restricted manner and may regulate the transcription of many genes.
Interleukin-23 is a key cytokine involved in the generation of Th17 effector cells. Clinical efficacy of an anti-p40 mAb blocking both IL-12 and IL-23 and disease association with single nucleotide polymorphisms in the IL23R gene raise the question of a functional role of IL-23 in psoriasis. In this study, we provide a comprehensive analysis of IL-23 and its receptor in psoriasis and demonstrate its functional importance in a disease-relevant model system. The expression of IL-23 and its receptor was increased in the tissues of patients with psoriasis. Injection of a mAb specifically neutralizing human IL-23 showed IL-23–dependent inhibition of psoriasis development comparable to the use of anti-TNF blockers in a clinically relevant xenotransplant mouse model of psoriasis. Together, our results identify a critical functional role for IL-23 in psoriasis and provide the rationale for new treatment strategies in chronic epithelial inflammatory disorders.
Psoriasis is an immune-mediated skin disorder that is inherited as a complex genetic trait. Although genome-wide association scans (GWAS) have identified 36 disease susceptibility regions, more than 50% of the genetic variance can be attributed to a single Major Histocompatibility Complex (MHC) locus, known as PSORS1. Genetic studies indicate that HLA-C is the strongest PSORS1 candidate gene, since markers tagging HLA-Cw*0602 consistently generate the most significant association signals in GWAS. However, it is unclear whether HLA-Cw*0602 is itself the causal PSORS1 allele, especially as the role of SNPs that may affect its expression has not been investigated. Here, we have undertaken an in-depth molecular characterization of the PSORS1 interval, with a view to identifying regulatory variants that may contribute to disease susceptibility. By analysing high-density SNP data, we refined PSORS1 to a 179 kb region encompassing HLA-C and the neighbouring HCG27 pseudogene. We compared multiple MHC sequences spanning this refined locus and identified 144 candidate susceptibility variants, which are unique to chromosomes bearing HLA-Cw*0602. In parallel, we investigated the epigenetic profile of the critical PSORS1 interval and uncovered three enhancer elements likely to be active in T lymphocytes. Finally we showed that nine candidate susceptibility SNPs map within a HLA-C enhancer and that three of these variants co-localise with binding sites for immune-related transcription factors. These data indicate that SNPs affecting HLA-Cw*0602 expression are likely to contribute to psoriasis susceptibility and highlight the importance of integrating multiple experimental approaches in the investigation of complex genomic regions such as the MHC.
Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.
Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.
Psoriasis is an inflammatory skin disorder that is inherited as a complex trait. Genetic studies have repeatedly highlighted HLA-C as the major determinant for psoriasis susceptibility, with the Cw*0602 allele conferring significant disease risk in a wide-range of populations. Despite the potential importance of HLA-C variation in psoriasis, either via an effect on peptide presentation or immuno-inhibitory activity, allele-specific expression patterns have not been investigated. Here, we used reporter assays to characterize two regulatory variants, which virtually abolished the response to TNF-α (rs2524094) and IFN-γ (rs10657191) in HLA-Cw*0602 and a cluster of related alleles. We validated these findings through the analysis of HLA-Cw*0602 expression in primary keratinocytes treated with TNF-α and IFN-γ. Finally, we showed that HLA-Cw*0602 transcripts are not increased in psoriatic skin lesions, despite highly elevated TNF-α levels. Thus, our findings demonstrate the presence of allele-specific differences in HLA-C expression and indicate that HLA-Cw*0602 is unresponsive to up-regulation by key pro-inflammatory cytokines in psoriasis. These data pave the way for functional studies into the pathogenic role of the major psoriasis susceptibility allele.
Human skin-resident IL-10+ regulatory dendritic cells induce T reg cells that suppress allogeneic skin graft inflammation.
Human skin immune homeostasis, and its regulation by specialized subsets of tissue-residing immune sentinels, is poorly understood. In this study, we identify an immunoregulatory tissue-resident dendritic cell (DC) in the dermis of human skin that is characterized by surface expression of CD141, CD14, and constitutive IL-10 secretion (CD141+ DDCs). CD141+ DDCs possess lymph node migratory capacity, induce T cell hyporesponsiveness, cross-present self-antigens to autoreactive T cells, and induce potent regulatory T cells that inhibit skin inflammation. Vitamin D3 (VitD3) promotes certain phenotypic and functional properties of tissue-resident CD141+ DDCs from human blood DCs. These CD141+ DDC-like cells can be generated in vitro and, once transferred in vivo, have the capacity to inhibit xeno-graft versus host disease and tumor alloimmunity. These findings suggest that CD141+ DDCs play an essential role in the maintenance of skin homeostasis and in the regulation of both systemic and tumor alloimmunity. Finally, VitD3-induced CD141+ DDC-like cells have potential clinical use for their capacity to induce immune tolerance.
Shwachman–Diamond syndrome (SDS) is a rare inherited disorder characterized by bone marrow (BM) dysfunction and exocrine pancreatic insufficiency. SDS patients have an increased risk for myelodisplastic syndrome and acute myeloid leukemia. Mesenchymal stem cells (MSCs) are the key component of the hematopoietic microenvironment and are relevant in inducing genetic mutations leading to leukemia. However, their role in SDS is still unexplored. We demonstrated that morphology, growth kinetics and expression of surface markers of MSCs from SDS patients (SDS-MSCs) were similar to normal MSCs. Moreover, SDS-MSCs were able to differentiate into mesengenic lineages and to inhibit the proliferation of mitogen-activated lymphocytes. We demonstrated in an in vitro coculture system that SDS-MSCs, significantly inhibited neutrophil apoptosis probably through interleukin-6 production. In a long-term coculture with CD34+-sorted cells, SDS-MSCs were able to sustain CD34+ cells survival and to preserve their stemness. Finally, SDS-MSCs had normal karyotype and did not show any chromosomal abnormality observed in the hematological components of the BM of SDS patients. Despite their pivotal role in the hematopoietic stem cell niche, our data suggest that MSC themselves do not seem to be responsible for the hematological defects typical of SDS patients.
Shwachman–Diamond syndrome; mesenchymal stem cells; bone marrow failure; SBDS
γδ T cells mediate rapid tissue responses in murine skin and participate in cutaneous immune regulation including protection against cancer. The role of human γδ cells in cutaneous homeostasis and pathology is poorly characterized.
In this study we show in vivo evidence that human blood contains a distinct subset of pro-inflammatory cutaneous lymphocyte antigen (CLA) and C-C chemokine receptor (CCR) 6 positive Vγ9Vδ2 T cells, which is rapidly recruited into perturbed human skin. Vγ9Vδ2 T cells produced an array of pro-inflammatory mediators including IL-17A and activated keratinocytes in a TNF-α and IFN-γ dependent manner.
Examination of the common inflammatory skin disease psoriasis revealed a striking reduction of circulating Vγ9Vδ2 T cells in psoriasis patients compared to healthy controls and atopic dermatitis patients. Decreased numbers of circulating Vγ9Vδ2 T cells normalized after successful treatment with psoriasis-targeted therapy. Together with the increased presence of Vγ9Vδ2 T cells in psoriatic skin, this data indicates redistribution of Vγ9Vδ2 T cells from the blood to the skin compartment in psoriasis.
In summary, we report a novel human pro-inflammatory γδ T cell involved in skin immune surveillance with immediate response characteristics and with potential clinical relevance in inflammatory skin disease.
IL-23 and Th17 cells are key players in tissue immunosurveillance and are implicated in human immune-mediated diseases. Genome-wide association studies have shown that the IL23R R381Q gene variant protects against psoriasis, Crohn's disease and ankylosing spondylitis. We investigated the immunological consequences of the protective IL23R R381Q gene variant in healthy donors. The IL23R R381Q gene variant had no major effect on Th17 cell differentiation as the frequency of circulating Th17 cells was similar in carriers of the IL23R protective (A) and common (G) allele. Accordingly, Th17 cells generated from A and G donors produced similar amounts of Th17 cytokines. However, IL-23-mediated Th17 cell effector function was impaired, as Th17 cells from A allele carriers had significantly reduced IL-23-induced IL-17A production and STAT3 phosphorylation compared to G allele carriers. Our functional analysis of a human disease-associated gene variant demonstrates that IL23R R381Q exerts its protective effects through selective attenuation of IL-23-induced Th17 cell effector function without interfering with Th17 differentiation, and highlights its importance in the protection against IL-23-induced tissue pathologies.
The nematode C. elegans displays complex dynamical behaviors that are commonly used to identify relevant phenotypes. Although its maintenance is straightforward, sorting large populations of worms when looking for a behavioral phenotype is difficult, time consuming and hardly quantitative when done manually. Interestingly, when submitted to a moderate electric field, worms move steadily along straight trajectories. Here, we report an inexpensive method to measure worms crawling velocities and sort them within a few minutes by taking advantage of their electrotactic skills. This method allows to quantitatively measure the effect of mutations and aging on worm's crawling velocity. We also show that worms with different locomotory phenotypes can be spatially sorted, fast worms traveling away from slow ones. Group of nematodes with comparable locomotory fitness could then be isolated for further analysis. C. elegans is a growing model for neurodegenerative diseases and using electrotaxis for self-sorting can improve the high-throughput search of therapeutic bio-molecules.
While there have been studies exploring regulatory variation in one or more tissues, the complexity of tissue-specificity in multiple primary tissues is not yet well understood. We explore in depth the role of cis-regulatory variation in three human tissues: lymphoblastoid cell lines (LCL), skin, and fat. The samples (156 LCL, 160 skin, 166 fat) were derived simultaneously from a subset of well-phenotyped healthy female twins of the MuTHER resource. We discover an abundance of cis-eQTLs in each tissue similar to previous estimates (858 or 4.7% of genes). In addition, we apply factor analysis (FA) to remove effects of latent variables, thus more than doubling the number of our discoveries (1,822 eQTL genes). The unique study design (Matched Co-Twin Analysis—MCTA) permits immediate replication of eQTLs using co-twins (93%–98%) and validation of the considerable gain in eQTL discovery after FA correction. We highlight the challenges of comparing eQTLs between tissues. After verifying previous significance threshold-based estimates of tissue-specificity, we show their limitations given their dependency on statistical power. We propose that continuous estimates of the proportion of tissue-shared signals and direct comparison of the magnitude of effect on the fold change in expression are essential properties that jointly provide a biologically realistic view of tissue-specificity. Under this framework we demonstrate that 30% of eQTLs are shared among the three tissues studied, while another 29% appear exclusively tissue-specific. However, even among the shared eQTLs, a substantial proportion (10%–20%) have significant differences in the magnitude of fold change between genotypic classes across tissues. Our results underline the need to account for the complexity of eQTL tissue-specificity in an effort to assess consequences of such variants for complex traits.
Regulation of gene expression is a fundamental cellular process determining a large proportion of the phenotypic variance. Previous studies have identified genetic loci influencing gene expression levels (eQTLs), but the complexity of their tissue-specific properties has not yet been well-characterized. In this study, we perform cis-eQTL analysis in a unique matched co-twin design for three human tissues derived simultaneously from the same set of individuals. The study design allows validation of the substantial discoveries we make in each tissue. We explore in depth the tissue-dependent features of regulatory variants and estimate the proportions of shared and specific effects. We use continuous measures of eQTL sharing to circumvent the statistical power limitations of comparing direct overlap of eQTLs in multiple tissues. In this framework, we demonstrate that 30% of eQTLs are shared among tissues, while 29% are exclusively tissue-specific. Furthermore, we show that the fold change in expression between eQTL genotypic classes differs between tissues. Even among shared eQTLs, we report a substantial proportion (10%–20%) of significant tissue differences in magnitude of these effects. The complexities we highlight here are essential for understanding the impact of regulatory variants on complex traits.
The skin immune system harbors a complex network of dendritic cells (DCs). Recent studies highlight a diverse functional specialization of skin DC subsets. In addition to generating cellular and humoral immunity against pathogens, skin DCs are involved in tolerogenic mechanisms to ensure the maintenance of immune homeostasis, as well as in pathogenesis of chronic inflammation in the skin when excessive immune responses are initiated and unrestrained. Harnessing DCs by directly targeting DC-derived molecules or selectively modulate DC subsets is a convincing strategy to tackle inflammatory skin diseases. In this review we discuss recent advances underlining the functional specialization of skin DCs and discuss the potential implication for future DC-based therapeutic strategies.
SALT, skin-associated lymphoid tissue; DCs, dendritic cells; AD, atopic dermatitis; CHS, contact hypersensitivity; DDCs, dermal DCs; MHC, major histocompatibility complex; pDCs, plasmacytoid DCs; TLR, toll-like receptor; SLE, systemic lupus erythematousus; HEL, hen egg lysozyme; iNOS, inducible nitric oxide synthase; TIP-DCs, TNF- and iNOS-producing DCs; NO, Nitric oxide; IDECs, inflammatory dendritic epidermal cells; NR-UVB, narrow-band UVB; PML, progressive multifocal leukoencephalopathy; TCI, transcutaneous immunization; Skin DCs; Homeostasis; Regulatory DCs; Chronic inflammation; Psoriasis
Skin ulcers are a dangerous and uncommon complication of vasculitis. We describe the case of a teenager suffering from Systemic Lupus Erythematosus with digital ulcer resistant to conventional therapy, treated successfully with Hyperbaric Oxygen Therapy. The application of hyperbaric oxygen, which is used for the treatment of ischemic ulcers, is an effective and safe therapeutic option in patients with ischemic vasculitic ulcers in combination with immunosuppressive drugs. Further studies are needed to evaluate its role as primary therapy for this group of patients.
Human skin and its immune cells provide essential protection of the human body from injury and infection. Recent studies reinforce the importance of keratinocytes as sensors of danger through alert systems such as the inflammasome. In addition, newly identified CD103+ dendritic cells are strategically positioned for cross-presentation of skin-tropic pathogens and accumulating data highlight a key role of tissue-resident rather than circulating T cells in skin homeostasis and pathology. This Review focuses on recent progress in dissecting the functional role of skin immune cells in skin disease.
The colon is a very rare metastatic localization. Here we report a case of colonic metastases from gastric adenocarcinoma whose clinical presentation was suggestive of a de novo adenocarcinoma of the ascending colon. The authors discuss that in the presence of a previous history of gastric cancer, immunohistochemical analysis on endoscopic biopsies may help in the definition of a differential diagnosis. Furthermore, this rare metastatic localization might suggest a poor prognosis and a more accurate diagnostic work-up.
Gastric adenocarcinoma; Colonic metastases; CDX-2
A number of studies in recent years have shown that members of the Roundabout (Robo) receptor family, Robo1 and Robo2, play significant roles in the formation of axonal tracks in the developing forebrain and in the migration and morphological differentiation of cortical interneurons. Here, we investigated the expression and function of Robo3 in the developing cortex. We found that this receptor is strongly expressed in the preplate layer and cortical hem of the early cortex where it colocalizes with markers of Cajal–Retzius cells and interneurons. Analysis of Robo3 mutant mice at early (embryonic day [E] 13.5) and late (E18.5) stages of corticogenesis revealed no significant change in the number of interneurons, but a change in their morphology at E13.5. However, preliminary analysis on a small number of mice that lacked all 3 Robo receptors indicated a marked reduction in the number of cortical interneurons, but only a limited effect on their morphology. These observations and the results of other recent studies suggest a complex interplay between the 3 Robo receptors in regulating the number, migration and morphological differentiation of cortical interneurons.
interneuron; morphology; Robo
Cell migration entails the dynamic redistribution of adhesion receptors from the cell rear toward the cell front, where they form new protrusions and adhesions. This process may involve regulated endo-exocytosis of integrins. Here we show that in primary neutrophils unengaged αL/β2 integrin (LFA-1) is internalized and rapidly recycled upon chemoattractant stimulation via a clathrin-independent, cholesterol-sensitive pathway involving dynamic partitioning into detergent-resistant membranes (DRM). Persistent DRM association is required for recycling of the internalized receptor because 1) >90% of endocytosed LFA-1 is associated with DRM, and a large fraction of the internalized receptor colocalizes intracellularly with markers of DRM and the recycling endocytic compartment; 2) a recycling-defective mutant (αL/β2Y735A) dissociates rapidly from DRM upon being endocytosed and is subsequently diverted into a late endosomal pathway; and 3) a dominant negative Rab11 mutant (Rab11S25N) induces intracellular accumulation of endocytosed αL/β2 and prevents its enrichment in chemoattractant-induced lamellipodia. Notably, chemokine-induced migration of neutrophils over immobilized ICAM-1 is abrogated by cholesterol-sequestering agents. We propose that DRM-associated endocytosis allows efficient retrieval of integrins, as they detach from their ligands, followed by polarized recycling to areas of the plasma membrane, such as lamellipodia, where they establish new adhesive interactions and promote outside-in signaling events.
We and others have shown that the minor, nonconserved allele Gln381 of the Arg381Gln single-nucleotide polymorphism (rs11209026G>A) of the IL-23 receptor gene (IL23R) protects against psoriasis. Moreover, we have recently shown impaired IL-23-induced IL-17A production and STAT-3 phosphorylation in Th17 cells generated in vitro from healthy individuals heterozygous for the protective A allele (GA). However, the biological effect of this variant has not been determined in homozygous carriers of the protective A allele (AA), nor in psoriatic patients. Here we expand our functional investigation of the IL23R Arg381Gln gene variant to include AA homozygous individuals. By using isolated memory CD4+ T cells, we found attenuated IL-23-induced Th17 response in heterozygous individuals. Moreover, we found that AA homozygous individuals were strikingly unresponsive to IL-23, with minimal or no IL-17A and IL-17F production and failure of human memory Th17 cell survival/expansion. Finally, IL-23-induced Th17 response was also attenuated in age- and sex-matched GA versus GG psoriatic patients undergoing systemic treatment. Taken together, our data provide evidence for an allele-dosage effect for IL-23R Gln381 and indicate that common gene alleles associated with complex diseases might have biological effects of considerable magnitude in homozygous carriers.