Impaired LAIR-1-mediated immune control due to collagen degradation in fibrosis.

Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.

'Nebbiolo' genome assembly allows surveying the occurrence and functional implications of genomic structural variations in grapevines (Vitis vinifera L.).

BACKGROUND: 'Nebbiolo' is a grapevine cultivar typical of north-western Italy, appreciated for producing high-quality red wines. Grapevine cultivars are characterized by possessing highly heterozygous genomes, including a great incidence of genomic rearrangements larger than 50 bp, so called structural variations (SVs). Even though abundant, SVs are an under-explored source of genetic variation mainly due to methodological limitations at their detection. RESULTS: We employed a multiple platform approach to produce long-range genomic data for two different 'Nebbiolo' clones, namely: optical mapping, long-reads and linked-reads. We performed a haplotype-resolved de novo assembly for cultivar 'Nebbiolo' (clone CVT 71) and used an ab-initio strategy to annotate it. The annotated assembly enhanced our ability to detect SVs, enabling the study of genomic regions not present in the grapevines' reference genome and accounting for their functional implications. We performed variant calling analyses at three different organizational levels: i) between haplotypes of clone CVT 71 (primary assembly vs haplotigs), ii) between 'Nebbiolo' and 'Cabernet Sauvignon' assemblies and iii) between clones CVT 71 and CVT 185, representing different 'Nebbiolo' biotypes. The cumulative size of non-redundant merged SVs indicated a total of 79.6 Mbp for the first comparison and 136.1 Mbp for the second one, while no SVs were detected for the third comparison. Interestingly, SVs differentiating cultivars and haplotypes affected similar numbers of coding genes. CONCLUSIONS: Our results suggest that SVs accumulation rate and their functional implications in 'Nebbiolo' genome are highly-dependent on the organizational level under study. SVs are abundant when comparing 'Nebbiolo' to a different cultivar or the two haplotypes of the same individual, while they turned absent between the two analysed clones.

CXCL4 drives fibrosis by promoting several key cellular and molecular processes.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

ZFP36 Family Members Regulate the Proinflammatory Features of Psoriatic Dermal Fibroblasts.

Dermal fibroblasts are strategically positioned underneath the basal epidermis layer to support keratinocyte proliferation and extracellular matrix production. In inflammatory conditions, these fibroblasts produce cytokines and chemokines that promote the chemoattraction of immune cells into the dermis and the hyperplasia of the epidermis, two characteristic hallmarks of psoriasis. However, how dermal fibroblasts specifically contribute to psoriasis development remains largely uncharacterized. In this study, we investigated through which cytokines and signaling pathways dermal fibroblasts contribute to the inflammatory features of psoriatic skin. We show that dermal fibroblasts from lesional psoriatic skin are important producers of inflammatory mediators, including IL-6, CXCL8, and CXCL2. This increased cytokine production was found to be regulated by ZFP36 family members ZFP36, ZFP36L1, and ZFP36L2, RNA-binding proteins with mRNA-degrading properties. In addition, the expression of ZFP36 family proteins was found to be reduced in chronic inflammatory conditions that mimic psoriatic lesional skin. Collectively, these results indicate that dermal fibroblasts are important producers of cytokines in psoriatic skin and that reduced expression of ZFP36 members in psoriasis dermal fibroblasts contributes to their inflammatory phenotype.

Troy/Tnfrsf19 marks epidermal cells that govern interfollicular epidermal renewal and cornification.

The skin epidermis is a highly compartmentalized tissue consisting of a cornifying epithelium called the interfollicular epidermis (IFE) and associated hair follicles (HFs). Several stem cell populations have been described that mark specific compartments in the skin but none of them is specific to the IFE. Here, we identify Troy as a marker of IFE and HF infundibulum basal layer cells in developing and adult human and mouse epidermis. Genetic lineage-tracing experiments demonstrate that Troy-expressing basal cells contribute to long-term renewal of all layers of the cornifying epithelium. Single-cell transcriptomics and organoid assays of Troy-expressing cells, as well as their progeny, confirmed stem cell identity as well as the ability to generate differentiating daughter cells. In conclusion, we define Troy as a marker of epidermal basal cells that govern interfollicular epidermal renewal and cornification.

ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections.

Sequencing the SARS-CoV-2 genome from clinical samples can be challenging, especially in specimens with low viral titer. Here we report Accurate SARS-CoV-2 genome Reconstruction (ACoRE), an amplicon-based viral genome sequencing workflow for the complete and accurate reconstruction of SARS-CoV-2 sequences from clinical samples, including suboptimal ones that would usually be excluded even if unique and irreplaceable. The protocol was optimized to improve flexibility and the combination of technical replicates was established as the central strategy to achieve accurate analysis of low-titer/suboptimal samples. We demonstrated the utility of the approach by achieving complete genome reconstruction and the identification of false-positive variants in >170 clinical samples, thus avoiding the generation of inaccurate and/or incomplete sequences. Most importantly, ACoRE was crucial to identify the correct viral strain responsible of a relapse case, that would be otherwise mis-classified as a re-infection due to missing or incorrect variant identification by a standard workflow.

Early and Long-Term Effects of Dupilumab Treatment on Circulating T-Cell Functions in Patients with Moderate-to-Severe Atopic Dermatitis.

Dupilumab, a mAb targeting IL-4 receptor alpha (IL-4Rα), markedly improves disease severity in patients with atopic dermatitis. However, the effect of IL-4Rα blockade on dynamics of circulating skin-homing T cells, which are crucial players in the pathologic mechanism of atopic dermatitis, has not been studied yet. In addition, it remains unknown whether dupilumab treatment induces long-lasting T- and B-cell polarization. Therefore, we studied the short- and long-term effects of dupilumab treatment on IL-4Rα expression and T-cell cytokine production within total and skin-homing (cutaneous lymphocyte antigen+/CCR4+) subpopulations in patients with moderate-to-severe atopic dermatitis. Dupilumab treatment completely blocked IL-4Rα expression and signal transducer and activator of transcription 6 phosphorylation in CD19+ B cells and CD4+ T cells within 2 hours of administration and through week 52. Although no change in the proportion of skin-homing T-cell subsets was found, dupilumab treatment significantly decreased the percentage of proliferating (Ki67+) and T helper type 2 and T helper type 22 cytokine-producing skin-homing CD4+ T cells at week 4. No evidence of general T helper type cell skewing following a year of dupilumab treatment was found. In summary, dupilumab treatment rapidly and stably inhibited IL-4Rα, which was accompanied by a strong early functional immunological effect specifically on skin-homing T cells without affecting overall T helper type cell skewing in the long term.

Confirmation of multiple endotypes in atopic dermatitis based on serum biomarkers.

BACKGROUND: Atopic dermatitis (AD) is a highly heterogeneous disease, both clinically and biologically, whereas patients are still being treated according to a "one-size-fits-all" approach. Stratification of patients into biomarker-based endotypes is important for future development of personalized therapies. OBJECTIVE: Our aim was to confirm previously defined serum biomarker-based patient clusters in a new cohort of patients with AD. METHODS: A panel of 143 biomarkers was measured by using Luminex technology in serum samples from 146 patients with severe AD (median Eczema Area and Severity Index = 28.3; interquartile range = 25.2-35.3). Principal components analysis followed by unsupervised k-means cluster analysis of the biomarker data was used to identify patient clusters. A prediction model was built on the basis of a previous cohort to predict the 1 of the 4 previously identified clusters to which the patients of our new cohort would belong. RESULTS: Cluster analysis identified 4 serum biomarker-based clusters, 3 of which (clusters B, C, and D) were comparable to the previously identified clusters. Cluster A (33.6%) could be distinguished from the other clusters as being a "skin-homing chemokines/IL-1R1-dominant" cluster, whereas cluster B (18.5%) was a "TH1/TH2/TH17-dominant" cluster, cluster C (18.5%) was a "TH2/TH22/PARC-dominant" cluster, and cluster D (29.5%) was a "TH2/eosinophil-inferior" cluster. Additionally, by using a prediction model based on our previous cohort we accurately assigned the new cohort to the 4 previously identified clusters by including only 10 selected serum biomarkers. CONCLUSION: We confirmed that AD is heterogeneous at the immunopathologic level and identified 4 distinct biomarker-based clusters, 3 of which were comparable with previously identified clusters. Cluster membership could be predicted with a model including 10 serum biomarkers.

CXCL4 Links Inflammation and Fibrosis by Reprogramming Monocyte-Derived Dendritic Cells in vitro.

Fibrosis is a condition shared by numerous inflammatory diseases. Our incomplete understanding of the molecular mechanisms underlying fibrosis has severely hampered effective drug development. CXCL4 is associated with the onset and extent of fibrosis development in multiple inflammatory and fibrotic diseases. Here, we used monocyte-derived cells as a model system to study the effects of CXCL4 exposure on dendritic cell development by integrating 65 longitudinal and paired whole genome transcriptional and methylation profiles. Using data-driven gene regulatory network analyses, we demonstrate that CXCL4 dramatically alters the trajectory of monocyte differentiation, inducing a novel pro-inflammatory and pro-fibrotic phenotype mediated via key transcriptional regulators including CIITA. Importantly, these pro-inflammatory cells directly trigger a fibrotic cascade by producing extracellular matrix molecules and inducing myofibroblast differentiation. Inhibition of CIITA mimicked CXCL4 in inducing a pro-inflammatory and pro-fibrotic phenotype, validating the relevance of the gene regulatory network. Our study unveils that CXCL4 acts as a key secreted factor driving innate immune training and forming the long-sought link between inflammation and fibrosis.

Leukocyte Associated Immunoglobulin Like Receptor 1 Regulation and Function on Monocytes and Dendritic Cells During Inflammation.

Inhibitory receptors are crucial immune regulators and are essential to prevent exacerbated responses, thus contributing to immune homeostasis. Leukocyte associated immunoglobulin like receptor 1 (LAIR-1) is an immune inhibitory receptor which has collagen and collagen domain containing proteins as ligands. LAIR-1 is broadly expressed on immune cells and has a large availability of ligands in both circulation and tissues, implicating a need for tight regulation of this interaction. In the current study, we sought to examine the regulation and function of LAIR-1 on monocyte, dendritic cell (DC) and macrophage subtypes, using different in vitro models. We found that LAIR-1 is highly expressed on intermediate monocytes as well as on plasmacytoid DCs. LAIR-1 is also expressed on skin immune cells, mainly on tissue CD14+ cells, macrophages and CD1c+ DCs. In vitro, monocyte and type-2 conventional DC stimulation leads to LAIR-1 upregulation, which may reflect the importance of LAIR-1 as negative regulator under inflammatory conditions. Indeed, we demonstrate that LAIR-1 ligation on monocytes inhibits toll like receptor (TLR)4 and Interferon (IFN)-α- induced signals. Furthermore, LAIR-1 is downregulated on GM-CSF and IFN-γ monocyte-derived macrophages and monocyte-derived DCs. In addition, LAIR-1 triggering during monocyte derived-DC differentiation results in significant phenotypic changes, as well as a different response to TLR4 and IFN-α stimulation. This indicates a role for LAIR-1 in skewing DC function, which impacts the cytokine expression profile of these cells. In conclusion, we demonstrate that LAIR-1 is consistently upregulated on monocytes and DC during the inflammatory phase of the immune response and tends to restore its expression during the resolution phase. Under inflammatory conditions, LAIR-1 has an inhibitory function, pointing toward to a potential intervention opportunity targeting LAIR-1 in inflammatory conditions.

Signal Inhibitory Receptor on Leukocytes-1 is highly expressed on lung monocytes, but absent on mononuclear phagocytes in skin and colon.

Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is expressed on human blood monocytes and granulocytes and inhibits myeloid effector functions. On monocytes, but not granulocytes, SIRL-1 expression is low or absent in individuals with the single nucleotide polymorphism (SNP) rs612529C. The expression of SIRL-1 in tissue and the influence of rs612529 hereon is currently unknown. Here, we used flow cytometry to determine SIRL-1 expression on immune cells in human blood and three barrier tissues; skin, colon and lung. SIRL-1 was expressed by virtually all neutrophils and eosinophils in these tissues. In contrast, SIRL-1 was not expressed by monocyte-derived cells in skin and colon, whereas it was highly expressed by lung classical monocytes. Lung monocytes from individuals with a rs612529C allele had decreased SIRL-1 expression, consistent with the genotype association in blood. Within the different monocyte subsets in blood and lung, SIRL-1 expression was highest in classical monocytes and lowest in nonclassical monocytes. SIRL-1 was not expressed by dendritic cells in blood and barrier tissues. Together, these results indicate that SIRL-1 is differentially expressed on phagocyte subsets in blood and barrier tissues, and that its expression on monocytes is genotype- and tissue-specific. Immune regulation of monocytes by SIRL-1 may be of particular importance in the lung.

Improved lipid productivity in Nannochloropsis gaditana in nitrogen-replete conditions by selection of pale green mutants.

BACKGROUND: Nannochloropsis gaditana is a photosynthetic unicellular microalgae considered one of the most interesting marine algae to produce biofuels and food additive due to its rapid growth rate and high lipid accumulation. Although microalgae are attractive platforms for solar energy bioconversion, the overall efficiency of photosynthesis is reduced due to the steep light gradient in photobioreactors. Moreover, accumulation of lipids in microalgae for biofuels production is usually induced in a two-phase cultivation process by nutrient starvation, with additional time and costs associated. In this work, a biotechnological approach was directed for the isolation of strains with improved light penetration in photobioreactor combined with increased lipids productivity. RESULTS: Mutants of Nannochloropsis gaditana were obtained by chemical mutagenesis and screened for having both a reduced chlorophyll content per cell and increased affinity for Nile red, a fluorescent dye which binds to cellular lipid fraction. Accordingly, one mutant, called e8, was selected and characterized for having a 30% reduction of chlorophyll content per cell and an almost 80% increase of lipid productivity compared to WT in nutrient-replete conditions, with C16:0 and C18:0 fatty acids being more than doubled in the mutant. Whole-genome sequencing revealed mutations in 234 genes in e8 mutant among which there is a non-conservative mutation in the dgd1 synthase gene. This gene encodes for an enzyme involved in the biosynthesis of DGDG, one of the major lipids found in the thylakoid membrane and it is thus involved in chloroplast biogenesis. Lipid biosynthesis is strongly influenced by light availability in several microalgae species, including Nannochloropsis gaditana: reduced chlorophyll content per cell and more homogenous irradiance in photobioreactor is at the base for the increased lipid productivity observed in the e8 mutant. CONCLUSIONS: The results herein obtained presents a promising strategy to produce algal biomass enriched in lipid fraction to be used for biofuel and biodiesel production in a single cultivation process, without the additional complexity of the nutrient starvation phase. Genome sequencing and identification of the mutations introduced in e8 mutant suggest possible genes responsible for the observed phenotypes, identifying putative target for future complementation and biotechnological application.

Dupilumab is very effective in a large cohort of difficult-to-treat adult atopic dermatitis patients: First clinical and biomarker results from the BioDay registry.

INTRODUCTION: Dupilumab has recently been approved for the treatment of moderate to severe atopic dermatitis (AD) in adults. Daily practice data on dupilumab treatment are scarce. OBJECTIVE: To study the effect of 16-week treatment with dupilumab on clinical response and serum biomarkers in adult patients with moderate-severe AD in daily practice. METHODS: Data were extracted from the BioDay registry, a prospective multicenter registry. Sixteen-week clinical effectiveness of dupilumab was expressed as number of patients achieving EASI-50 (Eczema Area and Severity Index) or EASI-75, as well as patient-reported outcomes measures (Patient-Oriented Eczema Measure, Dermatology Life Quality Index, Numeric Rating Scale pruritus). Twenty-one biomarkers were measured in patients treated with dupilumab without concomitant use of oral immunosuppressive drugs at five different time points (baseline, 4, 8, 12, and 16 weeks). RESULTS: In total, 138 patients treated with dupilumab in daily practice were included. This cohort consisted of patients with very difficult-to-treat AD, including 84 (61%) patients who failed treatment on ≥2 immunosuppressive drugs. At week 16, the mean percent change in EASI score was 73%. The EASI-50 and EASI-75 were achieved by 114 (86%) and 82 (62%) patients after 16 weeks of treatment. The most reported side effect was conjunctivitis, occurring in 47 (34%) patients. During dupilumab treatment, disease severity-related serum biomarkers (TARC, PARC, periostin, and IL-22), eotaxin-1, and eotaxin-3 significantly decreased. CONCLUSION: Treatment with dupilumab significantly improved disease severity and decreased severity-related serum biomarkers in patients with very difficult-to-treat AD in a daily practice setting.

Extracellular SPARC cooperates with TGF-ß signalling to induce pro-fibrotic activation of systemic sclerosis patient dermal fibroblasts.

OBJECTIVES: SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc. METHODS: Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-ß1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot. RESULTS: Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-ß and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-ß signalling. CONCLUSION: These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-ß dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.

Induction of Inflammation and Fibrosis by Semaphorin 4A in Systemic Sclerosis.

OBJECTIVE: To analyze the potential role of semaphorin 4A (Sema4A) in inflammatory and fibrotic processes involved in the pathology of systemic sclerosis (SSc). METHODS: Sema4A levels in the plasma of healthy controls (n = 11) and SSc patients (n = 20) were determined by enzyme-linked immunosorbent assay (ELISA). The expression of Sema4A and its receptors in monocytes and CD4+ T cells from healthy controls and SSc patients (n = 6-7 per group) was determined by ELISA and flow cytometry. Th17 cytokine production by CD4+ T cells (n = 5-7) was analyzed by ELISA and flow cytometry. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblast cells (n = 6) was analyzed by quantitative polymerase chain reaction, ELISA, Western blotting, confocal microscopy, and ECM deposition assay. RESULTS: Plasma levels of Sema4A, and Sema4A expression by circulating monocytes and CD4+ T cells, were significantly higher in SSc patients than in healthy controls (P < 0.05). Inflammatory mediators significantly up-regulated the secretion of Sema4A by monocytes and CD4+ T cells from SSc patients (P < 0.05 versus unstimulated SSc cells). Functional assays showed that Sema4A significantly enhanced the expression of Th17 cytokines induced by CD3/CD28 in total CD4+ T cells as well in different CD4+ T cell subsets (P < 0.05 versus unstimulated SSc cells). Finally, Sema4A induced a profibrotic phenotype in dermal fibroblasts from both healthy controls and SSc patients, which was abrogated by blocking or silencing the expression of Sema4A receptors. CONCLUSION: Our findings indicate that Sema4A plays direct and dual roles in promoting inflammation and fibrosis, 2 main features of SSc, suggesting that Sema4A might be a novel therapeutic target in SSc.

PD-1+CD8+ T cells are clonally expanding effectors in human chronic inflammation.

Chronic inflammatory diseases are characterized by recurrent inflammatory attacks in the tissues mediated by autoreactive T cells. Identity and functional programming of CD8+ T cells at the target site of inflammation still remain elusive. One key question is whether, in these antigen-rich environments, chronic stimulation leads to CD8+ T cell exhaustion comparable to what is observed in infectious disease contexts. In the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients, a model of chronic inflammation, an overrepresentation of PD-1+CD8+ T cells was found. Gene expression profiling, gene set enrichment analysis, functional studies, and extracellular flux analysis identified PD-1+CD8+ T cells as metabolically active effectors, with no sign of exhaustion. Furthermore, PD-1+CD8+ T cells were enriched for a tissue-resident memory (Trm) cell transcriptional profile and demonstrated increased clonal expansion compared with the PD-1- counterpart, suggesting antigen-driven expansion of locally adapted cells. Interestingly, this subset was also found increased in target tissues in other human chronic inflammatory diseases. These data indicate that local chronic inflammation drives the induction and expansion of CD8+ T cells endowed with potential detrimental properties. Together, these findings lay the basis for investigation of PD-1-expressing CD8+ T cell targeting strategies in human chronic inflammatory diseases.